REVIEW

Development of genetically modified citrus plants for the controlof citrus canker and huanglongbing

Juliana M. Soares1 & Sameena E. Tanwir1 & Jude W. Grosser1 & Manjul Dutt1

Received: 11 November 2019 /Accepted: 28 April 2020# Sociedade Brasileira de Fitopatologia 2020

AbstractCitrus cultivation is challenging due to the plethora of abiotic and biotic stresses faced by the crop. In recent years, production hasbeen severely affected by diseases such as citrus canker and huanglongbing (HLB). Disease management is hampered as there isno field resistance to these diseases in any of the important commercially planted varieties. Traditionally, conventional breedingapproaches have been applied for the improvement of the susceptible cultivars; however, this technique is laborious and timeconsuming. Genetic transformation of citrus allows for the rapid integration of novel genes into the plant’s genome to developdisease-resistant transgenic plants. Therefore, efforts have been made to utilize genetic engineering tools to develop geneticallymodified citrus that are resistant to citrus canker and HLB. This review summarizes the major achievements made in thedevelopment of citrus canker and HLB tolerance using transgenic technologies.

Keywords Citrus . Canker . Citrus greening . Huanglongbing . Transgenics

Introduction

The genusCitrus, which belongs to the Rutaceae family, containsplants that produce some of themost popular fruit commodities inthe world (Chen et al. 2019). Citrus fruits provide a high dietaryfiber content and a robust source of vitamin C along with manyother vitamins, amino acids, organic acids, and minerals (Silalahi2002; Ting 1980). Citrus cultivation is challenging because of theplethora of problems faced by the crop, including several bioticand abiotic stresses. The major challenges include Xanthomonascampestris pv. citri (Xcc) and Candidatus Liberibacter asiaticus(CLas), which are the etiological agents of citrus canker andHuanglongbing (HLB), respectively (Mendonça et al. 2017).Most of the citrus varieties are highly susceptible to both of thesebacterial diseases and the absence of a permanent cure forces thegrowers to invest significant time and resources into integrativepest and disease management strategies (Gottwald and Graham2014).

Citrus canker is widespread in citrus-growing areasthroughout the world (Gottwald et al. 2002). The symptoms

include leaf, stem, and fruit lesions surrounded by a dark orwater-soaked margin and a yellowish halo, and subsequentfruit drop. Infected trees display a steady decline in healthand fruit production. This leads to reduced yields of citrusfruits and massive economic losses (Gong and Liu 2013).Several commercially important varieties, such as sweet or-ange, grapefruit, key lime, and lemon, are known to be highlysusceptible to citrus canker disease (Gottwald et al. 1993).Traditionally, exclusion and eradication programs were usedto prevent and control citrus canker (Gottwald et al. 2001). InFlorida, this eradication based management approach hasbeen subject to considerable legal challenges and has influ-enced national and international trade disrupting the move-ment of fresh fruit (Gottwald et al. 2002). Citrus canker wasfirst reported in U.S. in 1912 and since then the dynamics ofthe disease has always been very complicated. Canker hasbeen officially eradicated several times only to reappear again.For example, the state of Florida declared citrus canker erad-icated in 1933, but it was again spotted in 1986. Later on, thestate was declared free of canker by 1994 and in 1997 thedisease re-emerged (Gottwald et al. 1993, 2002, 2001;Loucks 1934; Stall and Civerolo 1991). An alternative to erad-ication is the copper-based bactericides which are highly toxicto bacterial pathogens, relatively cheap, presents lowmamma-lian toxicity and its chemical stability and long residual pe-riods led to the wide spread usage for citrus canker

* Manjul Duttmanjul@ufl.edu

1 Citrus Research and Education Center, University of Florida, LakeAlfred, FL, USA

https://doi.org/10.1007/s40858-020-00362-9

/ Published online: 25 May 2020

Tropical Plant Pathology (2020) 45:237–250

management (Adaskaveg and Hine 1985; Cha and Cooksey1991; Gottwald et al. 2002; Leite Jr and Mohan 1990; Olsonand Jones 1983). However, the mode of action in this group ofchemicals is strictly preventive with no curative or systemicactivity, considering this, the integrated management mea-sures rely heavily on the planting of resistant varieties(Gottwald et al. 2002).

HLB, one of the oldest known bacterial diseases of citrus, istransmitted by the Asian citrus psyllid (ACP), which is a phloemsap-sucking insect (Bové 2006). Since it was first detected inBrazil in March 2004 and in Florida in August 2005 (Halbertet al. 2012; Teixeira et al. 2005), this disease has rapidly spread toall citrus-growing regions in the Americas and has become amajor concern (Hall et al. 2010). HLB symptoms include leafmottling and yellowing that crosses leaf veins, stunted trees, leafand fruit drop, twig dieback, and lopsided, small, and bitter-tasting fruits (Dagulo et al. 2010; Etxeberria et al. 2009;Gómez 2008). Severe infection eventually results in tree death(Hodges and Spreen 2012).

Because CLas strictly colonizes the phloem, the pathogenbecomes inaccessible to most pesticidal sprays and biologicalcontrols that target plant surfaces. Graft-based chemotherapyand trunk injections are some of the options that the growershave to reach the plant vascular system to deliver antibiotics(Hu et al. 2018; Zhang et al. 2014). However, often the HLBchemical control is mostly bacteriostatic rather than bacteri-cidal and is considered effective only as a short-term strategyrequiring continuous application of antibiotics to suppress dis-ease development (Aubert and Bové 1980; Yang et al. 2018).Besides the frequent applications being costly, the existenceof a potential risk of the emergence of antibiotic-resistant bac-teria and the lack of immediate approval from the USEnvironment Protection Agency or other regulatory agenciesto the application of technique in large scale by growers makethe use of chemical control in this method not viable/practical(Yang et al. 2018). For this reason, the two main current man-agement strategies for reducing HLB spread into groves arethe use of insecticidal spraying to target the insect vector andremoval of infected trees, which serve as inoculum source.Since the HLB pathogen incubation period is highly unpre-dictable, depending on variables such as time of year, envi-ronmental conditions, tree age, host species/cultivar, and hor-ticultural health, the symptom emergence might range frommonths to years after initial infection (Chiyaka et al. 2012;Gottwald 2010). Therefore, an asymptomatic tree could bealready actively transmitting the bacteria to ACP before visualsymptoms appear. This results in the abovementioned controlstrategies to have only a marginal effect on HLB control(Chiyaka et al. 2012; Lee et al. 2015).

To date, no field resistance has been developed in any ofthe susceptible varieties using traditional breeding techniquesfor either of these diseases. While several research groupsaround the world are making great progress using

conventional breeding approaches to develop HLB and cankertolerant plants, it may be several years before any acceptableHLB and canker resistant cultivar becomes popular with thegrowers (Stover et al. 2015).

Citrus has a variety of reproductive idiosyncrasies whichconfound attempts to generate bacterial disease-resistant cul-tivars through conventional breeding. These include a longjuvenile phase, self-incompatibility, polyembryony, heterozy-gosity, and parthenocarpy (Grosser et al. 1990). In addition,resistance traits that are present in wild species cannot beeasily introgressed into commercial cultivars because hybrid-ization also transfers undesirable agronomic traits to the prog-eny plants (Grosser et al. 2009). The genetic engineering ofcitrus plants is an attractive approach amongst scientists be-cause it can result in a faster development of disease-resistantcitrus (Sun et al. 2019). The genetic modification of plantsinvolves an intentional alteration to the plant genome throughthe insertion of DNA sequences. Resistance (R) genes, anti-bacterial proteins from plant or non-plant sources, proteinsrelated to systemic immunity, and artificial small or microRNAs, among others, are examples of putative sequences tobe used in citrus improvement (Mourgues et al. 1998; Niuet al. 2006; Wally and Punja 2010). An advantage of geneticengineering is that the source of the transgenes is not neces-sarily restricted to organisms belonging to the same species asthe target, enabling the exchange of genetic informationacross kingdoms (Dong and Ronald 2019).

Advancements in genetic transformation of plants allowthe integration of novel genes into the plant genome to devel-op disease-resistant transgenic plants. Numerous approacheshave been harnessed for the targeted genetic manipulation ofcitrus cultivars for disease resistance (Boscariol et al. 2006;Dominguez et al. 2000; Fagoaga et al. 2006). Many successfultransformation events that have resulted in commercially im-portant crops resistance to bacterial diseases have been per-formed using genes from different organisms (Azevedo et al.2006; Dutt et al. 2015; Fagoaga et al. 2001). For instance,genes encoding antimicrobial peptides (AMPs), which canserve as bactericidal agents, as well as genes responsible forthe synthesis of defense-associated proteins might beemployed as sources of transgene for enhancement of citrusresistance (Rocha Tavano and Vieira 2015). Recently, RNAinterference (RNAi) and CRISPR techniques have been addedto the disease management process. In this review, the variousgenetic engineering strategies being used to develop citrusplants resistant to canker and HLB are summarized.

Methods of producing genetically modifiedcitrus plants

Agrobacterium-mediated transformation of citrus usingAgrobacterium tumefaciens bacterium was initially reported

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by Kobayashi and Uchimiya (1989). The commonly usedepicotyl mediated transformation was developed by Mooreet al. (1992). The mechanism behind Agrobacterium-mediat-ed transformation involves the transfer of specific fragmentsof DNA from the T-DNA region of the bacteria’s tumor-inducing (Ti) plasmid into the host cell genome (Gelvin2003). This gene transfer is mediated by many virulence fac-tors known as vir genes, which are located in the vir region ofthe bacterium’s Ti plasmid. Citrus plants can be transformedwith Agrobacterium using several different explant sources,including juvenile epicotyl pieces, mature stem tissue, andembryogenic calluses. However, the recovery of a large pop-ulation of transgenic plants has been restricted by many fac-tors such as low transformation efficiency (Pena et al. 1995),numerous escapees despite using selection chemicals (Yanget al. 2000), and lack of an efficient regeneration protocol(Gutiérrez-E et al. 1997). The transformation efficiency de-pends on many factors such as the explant age and type, thepre-culturing and co-cultivation conditions, the strain ofAgrobacterium, and the cultivar used. Extensive researchhas resulted in the development of improved protocols forthe gene t ic modif ica t ion of c i t rus p lan ts us ingAgrobacterium (Cervera et al. 1998; Dutt and Grosser 2009,2010; Dutt et al. 2009). The nucellar seedling explant trans-formation that is commonly used is however restricted toseedy, polyembyonic scions and rootstocks. In contrast, theembryogenic callus system can work better for seedless poly-embryonic selections, as well as varieties that are more recal-citrant to Agrobacterium infection of stem pieces (Dutt et al.2018). Monoembryonic scions can currently be transformedusing the mature tissue transformation protocol (Cervera et al.1998, 2008).

In addition to the Agrobacterium-mediated transformationtechnique, other methods have been developed that do not relyon gene transfer using Agrobacterium. Protoplasts are plantcells that have had their protective cell wall removed eithermechanically or enzymatically (Grosser and Gmitter 1990).Protoplasts can be obtained from either embryogenic callusesor cell suspension cultures (Guo et al. 2005; Kaur et al. 2018).Protoplast transformation is a direct DNA integration methodthat relies on using either polyethylene glycol (PEG) (Fleminget al. 2000) or electroporation (Niedz et al. 2003). The PEG-mediated method results in the agglutination of protoplastsand the subsequent uptake of DNA via endocytosis(Cocking 1977). Using electroporation, the DNA migratesinto the cell via the application of an electric pulse of highfield strength that forms pores in the cell membrane (Chenet al. 2006). The only drawback of using protoplast transfor-mation in citrus is the low regeneration of transgenic plantsfrom the protoplast culture. This is due to the lack of an effi-cient selection process and protoplast-to-plant regenerationprotocol following transformation. In spite of it, protoplasttransformation is a practical and robust tool for the genetic

engineering of many cultivars that are recalcitrant toAgrobacterium transformation (Dutt et al. 2018). At present,protoplast transformation is also restricted to lines that makeembryogenic callus or suspension cultures (Fleming et al.2000; Niedz et al. 2003).

Particle bombardment is a relatively new direct DNAintegration technique for citrus (Wu et al. 2019, 2016). Inthis technique, gold or tungsten particles are coated withnaked DNA molecules and delivered into the cell by ahigh-pressure burst of helium gas (Klein et al. 1992).There are several factors that limit the transformation effi-ciency, including the delivery of an optimal quantity ofDNA while minimizing injury to the plant tissue, themicroprojectile size and density, the coupling of DNAand microprojectiles, and the pressure of helium at whichthe microprojectiles are launched (Kikkert et al. 2005). Theoptimization of these parameters is critical for obtaininghigh transformation efficiencies.

Engineering the plant’s defense mechanismsfor development of disease resistance

Due to their sessile nature, plants are unavoidably exposedto a variety of abiotic and biotic stresses. To cope withbiotic stresses, plants have evolved a highly sophisticatedimmune system. It is divided into pre-formed and inducibledefense mechanisms. The pre-formed immunity is com-posed of several layers of chemical and physical barriers,including a waxy cuticle, the cell wall, and the plasmamembrane. Meanwhile, the inducible defense system isbased on pathogen recognition by the host, which triggerslocal and systemic defense responses within the plant(Ponce de León and Montesano 2013; Serrano et al. 2014;Underwood 2012). In the last few years, transgenic plantswhich are less susceptible to phytopathogenic bacteria havebeen produced. Candidate genes have been identified fromdifferent organisms (both vertebrates and invertebrates) forsubsequent incorporation into the citrus plants. To obtaindisease-resistant citrus plants, it is necessary to understandthe multifaceted phytopathogen system and to utilize can-didate pathogen-targeting genes.

For plant immunity to occur, pathogen-associated mo-lecular patterns (PAMPs), which are various proteins orother molecules synthesized by pathogens, must activatethe innate plant immune responses (Zipfel and Robatzek2010). The PAMPs are recognized by pattern recognitionreceptors (PRRs) that are located on the plant cell surface.This recognition then activates defense-related pathways(Mishina and Zeier 2007). This is called PAMP-triggeredimmunity (PTI) (Fig. 1a). To suppress PTI, pathogenshave evolved a virulence mechanism that enables themto directly inject effectors into the host cell using a type

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III secretory system (T3SS), which avoids PRR recogni-tion (Deng et al. 2017). To counterattack, plants have alsoevolved and became capable of synthesizing R-proteinsthat directly or indirectly detect pathogen virulence fac-tors. These proteins enable plants to avoid successfulpathogen infection (Fig. 1b) (Jones and Dangl 2006). Inaddition to local defense, plants can activate systemic ac-quired resistance (SAR). This is a broad-spectrum mech-anism which involves the synthesis of chemical mobilesignals in tissues that have been locally infected by path-ogens (Enyedi et al. 1992). These signals travels through-out the phloem tissues triggering plant defense responsesin the uninfected portions of the plant (Durrant and Dong2004; Fu and Dong 2013). Thus, the activation of theplants’ own defense systems by engineering the plants tosynthesize R-proteins or defense-related signals, is anoth-er approach used for the improvement of citrus and othercrops (Beyer 2010; Borhan et al. 2010; Brueggeman et al.2002; Dutt et al. 2013; Fitch et al. 1992; Grosser et al.2009; Hennin et al. 2001; Kawchuk et al. 2001; Roy-Barman et al. 2006; Xiao et al. 2003).

Plants also employ anti-microbial peptides (AMPs) as adefense mechanism (Benko-Iseppon et al. 2010). AMPsare synthesized by most living species and are also fun-damental components of an organism’s innate immunesystem. These cationic molecules can interact with thenegatively charged pathogen membranes by changingtheir electrochemical potential and altering the membranepermeability, which eventually leads to cell death (Leiet al. 2019). Because of their antimicrobial activity, broadspectrum, and low cytotoxicity, AMPs could potentiallyreplace traditional antibiotics (Vlieghe et al. 2010).Although most research on AMPs is focused on their ac-tivity against medically important pathogens, plant bio-technologists have successfully used this system to genet-ically engineer important agronomic crops to resist several

pathogens (Goyal and Mattoo 2014; Yevtushenko andMisra 2012).

Potential strategies for the developmentof citrus canker and huanglongbingresistance in citrus plants

R-gene-mediated resistance

The concept of host–pathogen interactions was elucidatedmore than 50 years ago by Harold Flor with the gene-for-gene theory (Flor 1971). This theory is based on the premisethat for each avirulence (Avr) gene present in the pathogen,there is a corresponding resistance (R) gene in the host (Flor1971). Resistance genes and their homologs are often highlyconserved in the plant kingdom and activate similar steps insignaling defense-related pathways to trigger resistance inplants. The use of genetic engineering to express these genesfrom either the same genera or across genera, might also bebeneficial to citrus improvement programs (Rommens et al.1995; Thilmony et al. 1995).

Flagellin is a protein that is present in bacterial flagella.Flagellin is recognized as a PAMP by the R-geneFLAGELLIN RECEPTOR 2 (FLS2) (Fig. 2) (Zipfel et al.2004). A flagellin peptide, flg22, from Xcc (flg22Xcc) mediat-ed an increase in reactive oxygen species (ROS) and inducedthe expression of an early defense responsive gene in resistantcitrus genotypes ‘Nagami’ kumquat and ‘Sun Chu Sha’ man-darin. However, this effect was not observed in the susceptible‘Duncan’ grapefruit and ‘Navel’ sweet orange (Dalio et al.2017; Shi et al. 2015). In an attempt to increase resistance toXcc, Nicotiana benthamiana FLS2 was overexpressed in‘Hamlin’ sweet orange and Carrizo citrange. When comparedto non-transformed controls, the transgenic lines showed areduction in susceptibility to Xcc when challenged with

Fig. 1 Schematic representation of the locally induced plant defensemechanisms activated upon pathogen infection: (a) Pathogen-associatedmolecular patterns (PAMPs) from pathogens are recognized by hostextracellular pattern-recognition receptors (PRRs) to activate adownstream signaling cascade which culminates in PAMP-triggered

immunity (PTI). b To suppress PTI, bacterial pathogen acquired theability to produce effectors that are directly injected into plant cells,resulting in effector-triggered susceptibility (ETS). In turn, the host cellsevolved to synthesize R-proteins which can recognize the pathogeneffectors and trigger a defense termed effector-triggered immunity (ETI)

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flg22Xcc. The FLS2 citrus ortholog from sweet orange not onlypoorly recognized flg22Xcc but also exhibited very low homol-ogy to the known functional FLS2 isoforms. This suggestedthat FLS2 from canker-susceptible citrus varieties might lackthe domains necessary to enhance basal defense responses(Hao et al. 2016a).

Two homologs of citrus FLS2 (CiFLS2: FLS2–1 andFLS2–2) were identified using the citrus genomic database(Xu et al. 2013). These homologs were evaluated by Shiet al. (2016). FLS2–1 from grapefruit and mandarin, andFLS2–2 from kumquat, were transiently expressed in the sus-ceptible ‘Duncan’ grapefruit. The leaves of these grapefruitwere either treated with flg22Xcc or infected with Xcc.Grapefruit plants that were treated with an emptyAgrobacterium vector were used as the control plants. Forthe grapefruit plants expressing FLS2–1 (from mandarin)and FLS2–2 (from kumquat), 24 h after treatments with eitherflg22Xcc or Xcc, an increase in the expression of the defense-related genes, WRKY22, GST1, and EDS1 was observed (Shiet al. 2015). Additionally, a reduction in the citrus cankersymptoms was observed for these grapefruit 24 h after treat-ment (Shi et al. 2016). These results indicate that FLS2–1 andFLS2–2 are resistance genes that could potentially be integrat-ed into citrus improvement programs. These resistance genescould be used to generate cisgenic lines, which are lines thatare produced by incorporating genes from the same species orcross-compatible species, to enhance PTI and citrus cankerresistance in susceptible species (Shi et al. 2016).

Another R-gene that has often been utilized for its ability toincrease resistance in agronomic crops is the Xa21 from rice.This gene encodes a receptor kinase-like protein (Ronald et al.1992). Besides Xa21, there are several other rice R-genes thatencode kinase proteins and confer resistance againstXanthomonas oryzae (Sun et al. 2004). As Xa21 confers

broad-spectrum resistance to most races of X. oryzae and cit-rus canker is caused by a species of Xanthomonas, the use ofthis R-gene has been considered as a potential alternative toincrease resistance in citrus improvement programs (Mendeset al. 2010; Omar et al. 2007). In the citrus genome there are atleast 52 putative homologs that share 55 to 60% amino acididentity with the rice Xa21 gene (Deng and Gmitter 2003).However, to date, there are no reports of a citrus Xa21 homo-log enhancing resistance to Xcc. When the rice Xa21 gene wasoverexpressed in sweet orange cultivars ‘Hamlin’, ‘Natal’,and ‘Pera’, an increase in resistance to Xcc was detected30 days after inoculation. ‘Hamlin’ showed the most consis-tent tolerance among the transgenic lines evaluated and in allplants a significant reduction in susceptibility was observed(Mendes et al. 2010). The transgenic ‘W. Murcott’ mandarinoverexpressing Xa21 was tested for Xcc resistance. Xcc wascapable of inducing canker symptoms in all the transgeniclines, but the development of symptoms was significantlyslower in several of the transformed lines compared to thenon-transgenic plants (Omar et al. 2018). OverexpressingXa21 in ‘Anliucheng’ sweet orange also led to increased tol-erance to Xcc following infection (Ding-li et al. 2014).

The Xcc PthA4 effector, which is a AvrBs3 family-type IIIeffector, is one of the main transcriptional activator-like(TAL) effectors of Xcc (Dalio et al. 2017; Dunger et al.2012). The PthA4 effector is a key protein involved in therecognition of corresponding promoter sequences in citrusgenomes (Fig. 2). By recognizing these promoter sequences,PthA4 can induce the expression of susceptibility genes andlead to the development of canker symptoms (Al-Saadi et al.2007; Boch et al. 2009). In sweet orange (Citrus sinensis), thePthA4 effector targets the CsMAF1 protein (Soprano et al.2013). When CsMAF1 was silenced using RNAi, the synthe-sis of the three tRNAs, tRNAHis, tRNALeu, and tRNAThr,

Fig. 2 Defense mechanism in a citrus cell. (a-b) Representation of citruscells. Cell a is under non-stress condition while cell b is depicted afterbiotic stress. a FLS2 extracellular receptor, thionin, and linalool(C10H18O) antimicrobial peptides, all produced constitutively by thecell; cytoplasmic oligomer of NPR1 and NDR1 interacting with RIN4and RPS2. b Flagellin from Xcc (flg22Xcc) being recognized by FLS2,leading to the activation of early defense responsive genes. PthA4 and

AvrRpt2 effectors mediating suppression of PTI. PthA4 interacting withcomponents of citrus canker pathogenicity, named LOB1 gene andprotein MAF1. AvrRpt2 eliminating RIN4 upon proteolysis, releasingRPS2 for activation of defense. Pathogen infection increasing in SAlevels leading to the monomerization of NPR1, which moves to thenucleus, inducing PR1 transcriptional accumulation. Pathogen infectionresulting in accumulation of antimicrobial peptides thionin and linalool

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which are regulated by MAF1 in yeast and mammals, wassubstantially enhanced in comparison to in the non-transgenic plants (Cieśla et al. 2007; Pluta et al. 2001; Shoret al. 2010). Interestingly, a significant increase in canker le-sions and sizes were observed in the citrus CsMAF1 RNAilines, indicating that CsMAF1 acts as a negative regulator oftRNA and restricts the formation of canker in citrus plants.Conversely, in lines in which CsMAF1 was overexpressed,the plantlet growth decreased and there was significantstunting and yellowing of the shoots (Soprano et al. 2013).CsMAF1 protein levels decreased in sweet orange plants in-fected with Xcc. However, in sweet orange plants inoculatedwith a deletion mutant of Xcc pthA4 no development of thedisease symptoms was observed either in non-transgenicplants or CsMAF1 RNAi lines. Although CsMAF1 has beenproven to be an important player in Xcc pathogenicity, it ishighly homologous to the mammalian and yeast MAF1.Therefore, the use of this protein in citrus improvement pro-grams may raise many questions as to whether CsMAF1 maybind to the human MAF1/RNA Pol III complex. It may alsobe questioned whether PthA4 may interfere in the interactionbetween CsMAF1 and RNA Pol III (Soprano et al. 2013).

In citrus plants, the LATERAL ORGAN BOUNDARIES 1(CsLOB1) gene is a disease-susceptibility gene responsible forcanker susceptibility. The gene is triggered when the CsLOB1promoter makes contact with the PthA4-effector binding ele-ments (EBEs) (Hu et al. 2014). ‘Duncan’ grapefruit has twoalleles of the CsLOB1 gene because it is a product of hybridi-zation between pummelo and sweet orange (Jia et al. 2016; Wuet al. 2014; Xu et al. 2013). The CRISPR/Cas9 technique wasused to modify both the CsLOB1 gene and the promoter inefforts to modify the interaction between the PthA4 EBE andthe CsLOB1 promoter (Jia et al. 2016, 2017; Peng et al. 2017).Interestingly, when transgenic plants with a single genome-edited allele were challenged with the Xcc pathogen, the diseasesymptomswere not eliminated. A single allele modification wasunable to promote canker resistance. However, the pustulescaused by Xcc were much reduced when compared to the pus-tules observed in wild-type grapefruit (Jia et al. 2016). Thereason for this phenotype was that in the modified plants, thesusceptibility gene was still capable of being activated, but to amuch lower extent. Considering this, the activation of only asingle allele of susceptibility is needed to trigger susceptibility,and mutations in both alleles are necessary to acquire resistanceagainst citrus canker (Jia et al. 2016).

The complex interaction between R-protein and effector isoften regulated by an intermediate protein called guard pro-tein. The effector-guardee complex is recognized by the Rprotein to trigger resistance. The Arabidopsis NON-RACESPECIFIC DISEASE RESISTANCE 1 (NDR1) gene, whichis a positive regulator of salicylic acid (SA) accumulation, isrequired in the interaction between effector R-proteins such asRPM1, RPS2, and RPS5 (Fig. 2). Lu et al. (2013) identified a

citrus ortholog of NDR1 (CsNDR1) and tested it for comple-mentation of the ndr1 Arabidopsis mutant. The overexpres-sion of CsNDR1 enhanced the resistance of Arabidopsis toboth Pseudomonas syringae strains and Hyaloperonosporaarabidopsidis, which indicated that this gene might mediatebroad-spectrum disease resistance in plants. CsNDR1 in-creased SA biosynthesis and induced the expression of thePATHOGENESIS-RELATED PROTEIN 1 (PR1) gene.Altogether, CsNDR1 was considered to be a functionalortholog ofArabidopsis NDR1 and its overexpression in trans-genic citrus plants could potentially lead to enhanced resis-tance against citrus diseases (Lu et al. 2013).

Antimicrobial peptide (AMP)-mediated resistance

In plants, AMPs are synthesized against pathogens as part of afirst line of innate immunity (Veronese et al. 2003). Thisgroup of proteins includes several small peptides such as lipidtransfer proteins, plant defensins, and thionins (Benko-Iseppon et al. 2010). Stover et al. (2013) conducted a detailedin vitro evaluation of 40 putative AMPs that were predicted tosimultaneously inhibit the bacterial growth of CLas and Xcc(Stover et al. 2013). Because CLas cannot be cultured in vitro,two other bacteria belonging to the Alphaproteobacteria class,Sinorhizobium meliloti and Agrobacterium tumefaciens, wereused as surrogates in the system. The AMPs, of which con-centrations of 1 μM or less were sufficient to inhibit thegrowth of all three bacterial species, were tachyplesin I,SMAP-29, LL-37, and melittin from the horseshoe crab(Tachypleus tridentatus), sheep (Ovis aries), human (Homosapiens), and honeybee venom (Apis mellifera), respectively.Additionally, the synthetic AMPs, D4E1 and D2A21, werealso effective in inhibiting the bacterial growth (Stover et al.2013). D2A21 has been previously identified as one of theAMPs with the greatest potential for application in geneticengineering against various tree pathogens (Rioux et al.2000). In transgenic tobacco plants, D2A21 successfully in-duced resistance against P. syringae pv. tabaci. Therefore, thesame construct was introduced into citrus. The transgenicplants showed increased resistance against Xcc, but not CLas(Hao et al. 2017).

Another interesting group of AMPs that has been used forcitrus genetic engineering research is the insect-derived attacins(Mondal et al. 2012; Yi et al. 2014). Attacins belong to a classof AMPs with a molecular size of around 20 kDa (Hultmarket al. 1983). Attacins target the outer membrane of Gram-negative bacteria (Engström et al. 1984). They exist in bothbasic (A, B, C, D) and acidic (E, F) forms and are capable ofaltering membrane permeability and protein synthesis in bacte-rial cells (Fig. 3) (Carlsson et al. 1998; Hultmark et al. 1983).

Genetic transformation using attacin genes (att) have beensuccessfully carried out in apple (attA, attE) and pear (attE)trees and resulted in a reduction in susceptibility to fire blight

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(Erwinia amylovora [Burr]) (Ko et al. 2002; Reynoird et al.1999). To reduce the severity of citrus canker and HLB incitrus, several studies were conducted with geneticallyengineered plants expressing attacin A (attA) (Boscariolet al. 2006; Cardoso et al. 2010; Felipe et al. 2013; Tavanoet al. 2019). ‘Hamlin’ sweet orange was stably transformedwith an attA construct and citrus canker tolerance was evalu-ated in eight selected regenerated plants. In seven of the trans-genic plants, some degree of resistance to Xcc was observed(Boscariol et al. 2006). Other varieties of sweet orange such as‘Natal,’ ‘Pera,’ and ‘Valencia’ were also transformed withattA gene for a separate study and were evaluated for resis-tance to Xcc. Except for ‘Pera’, which is naturally more resis-tant to Xcc, all the other varieties demonstrated significantreduction in disease severity (Cardoso et al. 2010).Transgenic lines expressing attA transgene showed some lev-el of tolerance to HLB with lowered CLas titer estimated byqPCR. One of the lines also had fewer visual HLB symptomsin comparison to the other lines and control (Felipe et al.2013). Contrary to what was observed by Boscariol et al.(2006) and Cardoso et al. (2010) with canker tolerance, attAlines were unable to provide durable resistance to HLB. It wassuggested that this varied response may have been related tothe constitutive nature of the transgene expression. This mayhave worked favorably for defense against Xcc, which isapoplastic, compared to against HLB, which is localized tothe phloem (Boscariol et al. 2006; Cardoso et al. 2010; Felipeet al. 2013). However, when attA was expressed specificallyin the phloem, no significant differences were detected be-tween the CLas titers of transgenic and non-transgenic plants,either at six or twelve months after budwood infection withthe bacterium (Tavano et al. 2019). These results indicatedthat the attAAMP does not provide the same protective effectagainst HLB as with Xcc.

Several other AMPs have been used successfully to controlXcc. Dermaseptin (Der), a cationic AMP exhibits broad-spectrum activity against both Gram-positive and Gram-negative bacteria, fungi, yeasts, and protozoa (Coote et al.

1998; De Lucca et al. 1998; Hernandez et al. 1992; Navon-Venezia et al. 2002; Osusky et al. 2005; Rivero et al. 2012;Yaron et al. 2003). Der exhibited inhibitory effects upon atleast two strains of Xanthomonas, X. axonopodis pv. citri andX. campestris pv. campestris (Furman et al. 2013). Transgenictrees that were overexpressing Der and were challenged withXcc, demonstrated attenuation or absence of canker lesiondevelopment with up to a 40% size reduction in comparisonto non-transformed controls. Considering its wide range ofantimicrobial action, Der has the potential to be employedagainst both bacterial and fungal pathogen systems that affectcitrus. Another AMP with activity against several Gram-negative and a few Gram-positive bacteria is derived fromthe cercopin gene from the Chinese tasar moth (Jaynes et al.1993). Phloem-specific expression of a synthetic codon-optimized cercopin B gene in ‘Tarocco’ blood orange, sub-stantially inhibited the spread of CLas in comparison to con-trol plants (Zou et al. 2017). Sarcotoxin IA, an AMP obtainedfrom the flesh fly, Sarcophaga peregrina, has also beenshown to aid in imparting increased resistance to citrus canker(Kobayashi et al. 2017). However, it may be difficult to de-regulate transgenic plants expressing insect-derived AMPswithout first fully understanding their effect on the humanhealth (Bernauer 2016).

Plants also make their own AMPs (Baltzer and Brown2011). Among the many that are produced, defensins,thionins, lipid transfer proteins, cyclotides, snakins, andhevein-like proteins are synthesized as part of the plant’s de-fense mechanism. These AMPs may be employed in biotech-nological applications to enhance plant resistance againstpathogens (Nawrot et al. 2014). The thionin family of AMPsare composed of low molecular weight molecules of about5 kDa. The thionins are cysteine-rich, positively charged atneutral pH, and have antibacterial, antifungal, anticancer, andcytotoxic activities (Guzmán-Rodríguez et al. 2015; Nawrotet al. 2014). This family of AMPs function by opening poresin the pathogen cell membranes causing cellular leakage ofpotassium and calcium ions (Fig. 3) (Oard 2011). The

Fig. 3 Mode of action ofantimicrobial peptides that targetthe bacterial cell membrane fordisruption which results in celldeath

243Trop. plant pathol. (2020) 45:237–250

C. sinensis and C. clementine genomes have several thioninhomologs. However only one of them has a signal peptide anda mature form containing the acidic protein domain. The ma-ture form of this AMP is composed of 47 amino acids witheight conserved cysteine (Cys) residues. Transgenic Carrizorootstocks expressing a modified version of the thionin geneunder the expression of a double 35S promoter were pro-duced. These transgenic lines exhibited reduced canker symp-toms with a concomitant decrease in bacterial growth com-pared to non-transgenic plants when challenged with Xcc.When inoculated with HLB-infected budwood, the transgenicplants also had lower CLas titers in comparison to the con-trols. These findings indicate that the modified thionin mightbe a helpful AMP in the fight against both citrus bacterialdiseases (Hao et al. 2016b).

It has been demonstrated that the AMP linalool has antimi-crobial activity against Xcc (Shimada et al. 2014). Transgenic‘Hamlin’ sweet orange overexpressing the linalool synthasegene, CuSTS3–1, from the ‘Miyagawa Wase’ satsuma man-darin had a higher accumulation of linalool in its leaves andreduced susceptibility to Xcc, compared to non-transgenicplants. It was speculated that linalool might act not only as adirect antibacterial agent, but also as a signal molecule in-volved in triggering a non-host resistance response againstXcc. (Shimada et al. 2014, 2017).

Systemic acquired resistance

During a pathogen attack, at the infection site, a widerange of signals are generated by the plant. These signalsmove throughout the vascular system communicating tothe uninfected tissues of the plant about a potential dan-ger. The plants respond by mounting their armory of de-fense pathways, which protects them against secondaryinfections. This very well-known phenomenon is termedsystemic acquired resistance (SAR) and confers resistanceagainst a broad range of plant pathogens (Fig. 4) (Durrantand Dong 2004; Kuć 1987). SAR is a broad-spectrumform of plant resistance that takes advantage of the plant’sown immune system. The development of transgenicplants expressing SAR-inducing proteins has a potentialto genetically improve citrus plants (Dutt et al. 2016). TheNPR1 protein is present as oligomer in the cytoplasm andupon pathogen infection it is reduced to its monomericform and the monomers move to the nucleus where theyactivate PR1 gene expression (Fig. 2) (Dong 2004).

Several studies have focused on the role of NPR1 as atarget for developing citrus plants with tolerance to HLB andXcc. The studies have attempted to do this by either overex-pressing the Arabidopsis or citrus NPR1 gene. Transgenic‘Duncan’ and ‘Hamlin’ lines expressing the ArabidopsisNPR1 (AtNPR1) displayed an increased resistance to citruscanker (Zhang et al. 2010). These transgenic lines were also

assayed for tolerance to HLB under greenhouse conditions.Three transgenic lines consistently remained tolerant to HLBeven after many rounds of inoculation with CLas-infectedpsyllids (Robertson et al. 2018). In a separate study, Duttet al. (2016) generated sweet orange transgenic lines express-ing AtNPR1 either under the control of constitutive or phloemspecific (AtSUC2) promoters and planted them in a field sitewith high disease pressure. Several lines remained tolerant toHLB. Evaluation of the defense-related gene expression in theselected lines demonstrated higher PR1, PR2, and WRKY70expression (Dutt et al. 2016). Interestingly, when sweet or-ange plants that were overexpressing AtNPR1 were chal-lenged with Xcc, increases in the expression of EDS1 andPR2, but not PR1, were observed (Boscariol-Camargo et al.2016). The citrus NPR1 homolog was also overexpressed in‘Duncan’ grapefruit. Confirmed transgenic lines that werehighly expressing NPR1 transcripts were inoculated withXcc. All the transgenic lines evaluated possessed increasedresistance to citrus canker bacteria and had high levels ofchitinase 1 expression. This indicated that defense-relatedgenes were activated following upregulation of the citrusNPR1 homolog (Chen et al. 2013). Other SAR-inducinggenes evaluated in citrus include the hrpN gene obtained fromErwinia amylovora. Transcription of this gene results in theformation of a harpin protein (Wei et al. 1992). This proteincauses a hypersensit ive response and SAR whenoverexpressed in plants (Wei and Beer 1995). In transgenic‘Hamlin’ citrus lines overexpressing the hrpN gene and inoc-ulated with Xcc, the severity of the disease symptoms wasreduced by 79% (Barbosa-Mendes et al. 2009).

Fig. 4 Schematic representation of the systemic resistance activatedduring plant pathogen attack. Systemic acquired resistance (SAR)mobile signal(s) generated in the local infected tissues are translocatedthroughout the plant vascular system (phloem tissue). This protectsuninfected portions of the tree against secondary infections

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Transgrafting

Transgrafting is a potential technique that can be used inhorticulture crops resulting in the generation of non-transgenic fruit products. It involves the combination atransgenic rootstock with a non-transgenic scion(Haroldsen et al. 2012; Lev-Yadun and Sederoff 2001).In a transgrafted plant, the transgenic rootstock synthe-sizes both immobile transgene product (ITP), which isvery unlikely to be transported through the graft union,and mobile transgene products (MTP) that is capable ofcrossing the graft union (Song et al. 2015). Examples ofMTP are signal molecules which are involved in eitherlong- or short- distance transport include RNA (mRNA,siRNA, miRNA), peptides and protein (Liu et al. 2017;Luo et al. 2018; Zhao and Song 2014). This techniquehas been used in grapevine to detect the presence of theShiva-1 antimicrobial peptide in the non-transgenic scion.The antimicrobial peptide was produced in a transgenicrootstock and transmitted through the xylem sap to thenon-transgenic scion (Dutt et al. 2007). When wild typescions grafted onto transgenic rootstocks expressing ei-ther a chimeric antimicrobial protein (CAP) or apolygalacturonase inhibitory protein (PGIP) resulted inreduction of Pierce’s disease (PD) symptoms in field con-ditions under 7 years of evaluation (Dandekar et al.2019). Genetically modified citrus rootstocks expressinga synthetic cationic antimicrobial peptide were buddedwith non-transgenic scions and planted in a site with highHLB disease pressure. It was observed that the rate ofinfection was significantly slower than comparable nontransgenic rootstock-scion combinations (Bergey et al.2015).

Physical defense

In order to prevent the physical penetration of pathogens, plantscount with their primary mechanical and structural barriers in-cluding cuticle, hydrocarbon polymers, waxes, stomata, cell wall,plasma membrane, among others (Bhuiyan et al. 2009;Fernández et al. 2016; Zhang et al. 2018). Upon pathogens per-ception, one of the responses triggered by the plant’s immunesystem is the modification of the plant cell wall at the site ofinfection through deposition of papillae enriched in (1,3)-β-glu-can (callose) which acts as a physical barrier to slow pathogeninvasion (Aist 1976; Coll et al. 2011; Stone and Clarke 1992).

Callose is synthesized by multiple isoforms of CALLOSESYNTHASES (CalS) forming complexes in several differentlocations in response to developmental and environmentalcues, including biotic and abiotic stresses (Granato et al.2019; Stone and Clarke 1992). Under non-stress condition thispolymer is present on cell walls, root hair, spiral thickenings intracheids, sieve plates, pollen grains and pollen tubes (Chen

and Kim 2009; Stone and Clarke 1992). In the phloem sieveplates callose regulates size exclusion limit of plasmodesmataconnections limiting the cell-to-cell movement of molecules(Ellinger and Voigt 2014). Callose deposition reduces theopen space of the pores in the sieve plate and resulted inplugging of the HLB-infected flush (Achor et al. 2020).

To investigate the effect of CalS in the morphology ofC. limon leaves, Enrique et al. (2011) silenced this enzymeand evaluate the phenotype upon pathogen infection. Thepathogen used in these assays were Xcc. Different from wildtype plants, in silenced lines (RNAiCalS1) inoculated withXcc no callose deposition was observed forty-eight hours afterinfection (a.i). In the RNAi CalS1 lines canker symptomswere observed to be slightly stronger after 10 days a.i. whencompared to wild-type plants. This indicated that plant cellwall-associated defense is the initial barrier againstXanthomonas infection in lemon (Enrique et al. 2011).

Future directions

Bacterial disease management through the genetic engineer-ing of plants is a reasonable alternative to traditional plantbreeding for citrus canker control. There are many candidategenes that have shown potential and are highly efficient inreducing the disease symptoms. However, the number ofgenes that can reduce HLB infection is relatively small andmost of them do not seem to be fully capable of solving theHLB crisis that the citrus industry is facing. HLB is the biggestthreat ever encountered by citrus growers, and therefore acombination of management tactics that can efficiently tacklethis disease is promptly needed. A transgenic solution to adisease is different in a long-term perennial tree crop than anannual crop, so any commercialization of a transgenic solutionshould include at least two sources of strong tolerance/resis-tance, as necessary to prevent a mutation in the pathogen fromovercoming the resistance. The two or more transgenes shouldwork by different mechanisms to effectively back each otherup – thus preventing a mutant strain of the pathogen fromtaking over in the field. It is possible that the gene stackingto produce transgenic plants withmultiple genes, coupled withcultural, chemical, and quarantine measures might aid citrusplants to coexist with HLB. Novel techniques such asCRISPR-mediated genome editing, which can be used to pre-cisely edit the genome with or without putative resistancegene stacking, is also an attractive technique to be includedin the integrated disease management package. These tech-niques can be less labor intensive and less time consumingcompared to conventional methods of disease control.

Compliance with ethical standards

Conflict of interest On behalf of all authors, the corresponding authorstates that there is no conflict of interest.

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