Transient expression of pepper Bs2 gene in ... - Wiley Online Library

Plant Pathology (2012) 61, 648–657

Doi: 10.1111/j.1365-3059.2011.02558.x

Transient expression of pepper Bs2 gene in Citrus limon as an approach to evaluate its utility for management of citrus canker disease L. N. Sendıńa, M. P. Filipponea, I. G. Orcea, L. Riganob, R. Enriquec, L. Penãd, A. A. Vojnovb, M. R. Maranoc and A. P. Castagnaroa* a

Seccioń Biotecnologıá de la Estacioń Experimental Agroindustrial Obispo Colombres (EEAOC) –Unidad Asociada al Instituto Superior de Investigaciones Biolo´gicas (INSIBIO; CONICET-UNT), Av. William Cross 3150, CP: T4101XAC, Las Talitas, Tucumań; b Fundacioń Pablo Cassara´, Centro de Ciencia y Tecnologıá ‘Dr. Cesar Milstein’, Saladillo 2468, CP: C1440FFX, Ciudad de Buenos Aires; cInstituto de Biologıá Molecular y Celular de Rosario (IBR-CONICET), A´rea Virologıá, Facultad de Ciencias Bioquı´micas y Farmaceúticas, Universidad Nacional de Rosario, Suipacha 531, CP: S2002LRK, Rosario, Argentina; and dDpto. Proteccioń Vegetal y Biotecnologıá, Instituto Valenciano de Investigaciones Agrarias (IVIA), Apartado Oficial, 46113-Moncada, Valencia, Spain

The pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains containing the avrBs2 avirulence gene in susceptible pepper and tomato. The avrBs2 gene is highly conserved in the Xanthomonas genus and when bacteria lack this gene their growth in a susceptible host is diminished, indicating that the avrBs2 gene product could confer an adaptive advantage to the pathogen. The avrBs2 of Xanthomonas citri subsp. citri (Xcc), cause of citrus canker, shares 96% homology with avrBs2 of Xcv. To evaluate if Bs2 could recognize avrBs2 of Xcc in citrus plants and thereby activate plant defence mechanisms to increase resistance to canker, transient expression experiments were conducted using Agrobacterium tumefaciens in lemon plants subsequently challenged with wildtype Xcc. The results showed that transient expression of Bs2 reduced canker formation in lemon and induced plant defence mechanisms, as shown by callose deposition and PR-1 expression. Moreover, when an avrBs2 mutant of Xcc was used, no decrease in disease symptoms was observed. This work shows that the Bs2 gene from Solanaceae is functional in lemon, a member of the Rutaceae family. Therefore, Bs2 is a potential candidate gene for stable expression in transgenic citrus plants in order to improve resistance to canker disease. Keywords: avrBs2 avirulence gene, citrus breeding, defence response, disease resistance

Introduction Citrus canker is a serious bacterial disease that affects most commercial citrus species and varieties. Xanthomonas citri subsp. citri (Xcc), causal agent of this disease, produces pustule-like cankers and necrotic lesions on leaves, stems and fruits (Brunings & Gabriel, 2003; Das, 2003). In severe attacks, it causes defoliation and fruit quality loss, which results in reduced production. It is thought that citrus canker first appeared in Southeast Asia and India but is now present in Japan, South and Central Africa, the Middle East, the Pacific Islands, South America and the USA (Das, 2003). Although some areas of the world have eradicated citrus canker and others have ongoing eradication programmes, the disease remains endemic in most regions where it has appeared

*E-mail: [email protected]

Published online 30 November 2011

648

(Das, 2003). Because of its rapid spread, high potential for damage and impact on export sales and domestic trade, citrus canker is a significant threat to all citrusgrowing countries. Citriculture is one of the most important agro-industrial activities in Argentina, one of the main lemon producers in the world (Federacioń Argentina del Citrus, 2009). The citrus industry in Argentina depends heavily on fruit export; thus quarantine diseases, such as citrus canker and black spot, are of vital economic importance. Attempts to minimize the impact of phytosanitary problems in citrus plants have been made through conventional breeding procedures. However, these procedures have important limitations because most species are apomictic, have high heterozygosis and long juvenile periods (Herrero et al., 1996; Penã & Navarro, 1999). Genetic transformation is therefore an attractive technology in order to improve citrus fruit production, as it can overcome most of the limitations found in conventional breeding mentioned above. Genetic transformation and recovery of transgenic citrus trees have been achieved in various species, hybrids and Citrus relatives such as rough ª 2011 The Authors Plant Pathology ª 2011 BSPP

Bs2 gene expression for management of citrus canker

lemon, sweet orange, Carrizo citrange, Poncirus trifoliata and Mexican lime (Penã & Navarro, 1999). However, some citrus species such as Citrus limon remain recalcitrant to regeneration and transformation. One of the strategies previously used to improve citrus canker resistance through genetic transformation is the introduction of genes that encode antimicrobial peptides, which control the pathogen in a direct way. For example, attacin A, an antibacterial peptide from Tricloplusia ni, was expressed in Citrus sinensis cv. Hamlin (Boscariol et al., 2006) and sarcotoxin, a potent bactericidal peptide from Sarcophaga peregrine, was expressed in C. sinensis cv. Pera (Bespalhok Filho et al., 2001). In both cases, a reduction in citrus canker disease development could be observed. However, the use of antimicrobial peptides in plant tissues could exert a high selective pressure on the pathogen and thereby generate resistance (Yeaman & Yount, 2003; Fehri et al., 2005). Another strategy, without causing high selective pressure, is the use of genes that induce the plant’s own defence mechanisms, such as genes encoding elicitor compounds which activate general mechanisms against a broad spectrum of pathogens (Barbosa-Mendes et al., 2009) or genes that recognize pathogen-specific products, such as resistance genes (R-genes) which interact with a pathogen avirulence gene product (avr-gene) (Flor, 1971). R-gene products belong to one of a few classes of proteins with conserved modular structures (Hammond-Kosack & Jones, 1997; Martıńez Zamora et al., 2008), suggesting that homologous R-genes and the signalling pathways in which they participate may be widely conserved in the plant kingdom. For example, the Xa21 resistance gene from the wild species Oryza longistaminata confers a broad-spectrum resistance to most races of Xanthomonas oryzae pv. oryzae, responsible for bacterial blight in rice (Ronald et al., 1992). Mendes reported that Xa21 R-gene transgenic C. sinensis plants showed enhanced resistance to citrus canker (Mendes et al., 2010), indicating that this gene from rice is functional in a citrus genotype. However, it was observed that the effectiveness of the Xa21 gene in controlling the citrus canker was influenced by the citrus cultivar genetic background and it was not possible to identify transgenic lines with complete resistance to the pathogen (Mendes et al., 2010). In this study, interest is focused on the genetic interaction between the Bs2 resistance gene isolated from an isogenic line of pepper cv. Early Calwonder (ECW-20R Bs2 ⁄ Bs2) (Tai et al., 1999) and the avrBs2 avirulence gene from Xanthomonas campestris pv. vesicatoria (Xcv). The stable expression of the Bs2 gene can confer resistance in susceptible pepper, tomato and Nicotiana benthamiana against pathogenic strains of Xcv harbouring the corresponding avirulence gene (avrBs2) (Tai et al., 1999). Bacteria lacking the avrBs2 gene show a decreased growth of 10–100 fold in a susceptible host, as compared with bacteria harbouring the avirulence gene (Kearney & Staskawicz, 1990; Swords et al., 1996), indicating that this gene gives an adaptive Plant Pathology (2012) 61, 648–657

649

advantage to the pathogen. This feature of the avrBs2 gene and the fact that it is highly conserved in other species belonging to the Xanthomonas genus, suggests that Bs2 can recognize avrBs2 genes from different Xanthomonas species, and is therefore a promising candidate for generating resistance to diseases caused by bacteria of this genus. Taking this into account, and because of the high homology (96%) between the avrBs2 of Xcc and the avrBs2 of Xcv, this study looked at whether the Bs2 gene introduced into C. limon plants would recognize avrBs2 of Xcc and thereby activate defence mechanisms in the plant, generating improved resistance to citrus canker. Based on the aforementioned reasons, and considering that C. limon is still recalcitrant to genetic transformation, transient expression assays were used to evaluate the effect of Bs2 gene expression on development of canker symptoms in lemon plant leaves.

Materials and methods Plant material Buds were collected from pathogen-free lemon plants, C. limon cv. Eureka Frost Nuclear, grown at the Sanitation Center at EEAOC, Argentina. Collected buds were grafted on Cleopatra Mandarin (Citrus reshni) seedlings and grown for 6–12 months in a greenhouse (18–27C, 16 h light photoperiod, 70% RH). Young leaves from the resulting grafted lemon plants were used for all transient expression experiments. For assays with pepper plants, seedlings of Capsicum annuum cv. Margarita were grown at 23–25C with a photoperiod of 18 h.

Bacterial strains and culture conditions To reproduce canker disease under controlled growth conditions, plant leaves were inoculated with an Xcc isolate (wildtype strain) from lemon leaves with canker symptoms, obtained and identified in the Phytopathology laboratory at EEAOC. Strains in which the avrBs2 gene was disrupted were created by using the plasmid pKmobsacB (Quandt & Hynes, 1993). A 1259 bp fragment of the avrBs2 gene (XAC0076) was amplified by PCR using Xcc chromosomal DNA as a template and the primers avrBs2-sense (5¢-TCAAGAAACGCTGTTCTAGC-3¢) and avrBs2antisense (5¢-AATCACCAACGGCATTTCAC-3¢). The resulting amplicon was cloned into the pGEM T-Easy vector (Promega) according to the manufacturer’s instructions. The identity of the cloned fragment was confirmed by DNA sequencing. The 2 kb X fragment cassette conferring spectinomycin resistance from pHP45 X (Prentki & Krisch, 1984) was ligated as a HindIII fragment into pGEM T-Easy digested with HindIII within the avrBs2 gene. The resulting avrBs2: X allele was cloned as a 3259 bp EcoRI fragment into the pKmobsacB suicide vector digested with EcoRI, and later transferred by electroporation to Xcc.

650

L. N. Sendıń et al.

Spectinomycin-resistant colonies were selected, and double recombinants were selected on peptone-yeast extract-malt extract (PYM) nutrient medium containing 5% sucrose and spectinomycin. The insertion of the X allele in the avrBs2 gene was confirmed by PCR and Southern blot. All Xcc strains (wild type and mutant strains) were cultured at 28C in PYM nutrient medium, supplemented with D-glucose at a final concentration of 1Æ5% (w ⁄ v) at 28C for 24 h.

Plasmid constructions pLS: the pMOG180 plasmid expression cassette is composed of the 35S promoter of Cauliflower mosaic virus (CaMV) with double enhancer and the Alfalfa mosaic virus (AMV) RNA 4 leader sequence, a BamHI restriction site and the nopaline synthase gene (nos) terminator. To obtain a negative control plasmid for gene transient expression analysis, the cassette was digested and cloned into the pCAMBIA 2301(Clontech) plant transformation vector at the EcoRI and HindIII sites. The resulting plasmid was named pLS (Fig. 1). pLS-Bs2: the full-length ORF of the Bs2 gene from pepper cv. ECW-20R (Tai et al., 1999), was amplified by PCR from a transformation vector (35S-Bs2 construct; Tai et al., 1999) harbouring the Bs2 gene (kindly supplied by Dr Staskawikz, University of California, Berkeley) by using the Bs2-forward (5¢-ATC AGG ATC CTA TGG CTC ATG CAA GTG T-3¢) and Bs2-reverse (5¢-TAT GGA TCC CAA GAG TTC AAT CCT TC-3¢) primers, both of which carry BamHI sites (as indicated in italic type) at their 5¢ ends. The resulting amplification product was digested and subcloned into the BamHI site of pLS.

The resulting plasmid, pLS-Bs2, harboured the Bs2 gene under the control of the 35S promoter from CaMV with double enhancer, the AMV RNA 4 leader sequence, and the nos terminator. pLS also contains the 35S ⁄ gusA ⁄ nos cassette, which serves as a reporter gene, and the nos ⁄ nptII ⁄ nos cassette, used as a selectable marker gene (Fig. 1). 35S-avrBs2: the plasmid consists of the avrBs2 ORF from Xcv (Swords et al., 1996) cloned between the 35S CaMV promoter and the nopaline synthase 3¢ sequences of pMD1, a derivative of pBI121 (Clontech) in which the GUS reporter gene (also known as uidA) has been replaced by a synthetic polylinker (kindly supplied by Dr Staskawikz, University of California, Berkeley). The three plasmids used in transient expression assays (pLS, pLS-Bs2 and 35S-avrBs2) were mobilized into Agrobacterium tumefaciens strain EHA105 by electroporation.

Agrobacterium infiltration Individual Agrobacterium colonies were grown for 20 h in 5 mL Luria–Bertani broth (LB) medium at 28C. A 0Æ5 mL aliquot of this suspension was used to inoculate a 50 mL culture (LB broth with: 20 lM acetosyringone, 50 mg L)1 kanamycin, 25 mg L)1 nalidixic acid and 10 mM MES pH 5Æ7), which was grown for 16–20 h at 28C. Bacteria were pelleted by centrifugation, resuspended in plant infiltration medium (10 mM MgCl2, 10 mM MES pH 5Æ7 and 150 lM acetosyringone) to an optical density of 0Æ5 (600 nm), and incubated at 21C for a minimum of 3 h (Llave et al., 2000). The Agrobacterium suspension was injected into the underside of leaves

(a) T-nos

gusA

CaMV 35S 2x

Right-border

Bs2

T-nos

CaMV 35S 2x

AMVRNA 4

HindIII BamHI

CaMV 35S

nptII

Left-border

EcoRI

BamHI

CaMV 3´PolyA

(b) T-nos

gusA

CaMV 35S 2x

Right-border

T-nos

HindIII

AMVRNA 4

CaMV 35S 2x

BamHI

CaMV 35S

nptII

CaMV 3´PolyA

Left-border

EcoRI

(c) P-nos

Right-border

nptII

T-nos

CaMV 35S

avrBs2

T-nos

Left-border

Figure 1 Schematic representation of genetic construction T-DNAs. (a) pLS-Bs2: The Bs2 gene is controlled by the doubly enhanced Cauliflower mosaic virus promoter (CaMV 35S 2·), the Alfalfa mosaic virus RNA 4 leader sequence (AMV-RNA 4) and the nopaline synthase terminator (T-nos). The Bs2 cassette is flanked by the neomycin phosphotransferase II gene (nptII) between the CaMV 35S 2· promoter and the CaMV 3¢ UTR terminator (CaMV 3¢ PolyA) and by the gusA gene between the CaMV 35S promoter and the T-nos. Each gene transcription orientation is indicated by arrows, and EcoRI, BamHI and HindIII restriction sites are marked with vertical lines; (b) pLS: this construction is the same as above but without the Bs2 gene; and (c) 35S-avrBs2 plasmid: consists of the avrBs2 ORF cloned between the CaMV 35S promoter and T-nos of pMD1 (B. Savidge, unpublished results).



through a little incision using a 3 mL syringe. Young lemon leaves (50–80% expanded) were used for all transient expression assays. In order to determine the effect of Bs2 gene expression on citrus canker symptoms, zones of leaves were infiltrated with a mixture of an equal volume of Agrobacterium suspension containing the Bs2 construction (pLSBs2) and the avrBs2 construction (35S-avrBs2 plasmid), while other zones were infiltrated with an Agrobacterium suspension containing the Bs2 construction alone or with the negative control plasmid (pLS). For plant defence marker assays, leaves were infiltrated either with the Bs2 expression construct or the negative control. Ten young leaves per plant and three plants per treatment were used. All infiltrated plants were placed in the greenhouse at 21C for 24 h prior to Xcc inoculation. All experiments were carried out in triplicate.

Xcc inoculation For Xcc inoculation, three different methods previously described by other authors (Siciliano et al., 2006) were used: (i) nicking the underside of leaves with a razor blade and subsequent spraying of the bacteria, (ii) pressure infiltrating the bacteria with a 3 mL syringe without a needle, and (iii) application of a bacterial suspension by using a cotton swab. The last method mimics a natural Xcc infection process, in which bacteria enter leaves through natural openings (stomata), followed by colonization of the apoplast. The bacterial culture was pelleted by centrifugation and resuspended in 10 mM MgCl2 to a concentration of 108 CFU mL)1 for inoculation methods 1 and 3, and to a concentration of 105 CFU mL)1 for method 2. Inoculated plants were kept in a growth cabinet at 25– 28C, high humidity (70% RH), and a photoperiod of 16 h light with an intensity of 150–200 lE m)2 s)1. Disease progression was monitored both phenotypically and by bacterial population growth analysis. In the case of inoculation method (i), the evaluation was carried out by determining bacterial growth (see below). When inoculation methods (ii) and (iii) were used, the average canker number per cm2 of leaf inoculated was calculated. For hypersensitive response (HR) tests on pepper plants, the agroinfiltrations and Xcc inoculation were carried out by infiltration into young leaves with needleless syringes.

Xcc population on leaves inoculated by nicking and spraying Two weeks after inoculation, Xcc was isolated from leaves according to the method described by Siciliano et al. (2006). Three 1 cm2 leaf disks were selected randomly from inoculated leaves and immersed in 500 lL of 10 mM MgCl2 into a 2 mL Eppendorf tube. Bacterial cells were collected by homogenization of the plant tissue using a plastic pestle. The corresponding suspension was stirred at room temperature for 1 min and serial dilutions Plant Pathology (2012) 61, 648–657

651

of the suspension were plated on three Petri dishes containing PYM agar medium, and the bacterial growth was reported as CFU cm)2 of inoculated leaf.

Determination of callose deposits Callose is a b-1,3 glucan with (1,6) modifications that can be synthesized in plants as a response to mechanical wounds, physiological stresses, and pathogen attacks (Stone & Clarke, 1992). To see if callose formation is one of the mechanisms involved in plant defence against Xcc, callose deposition was assessed in plants infiltrated with the pLS-Bs2 or the pLS vectors and inoculated with wildtype Xcc or the avrBs2 mutant of Xcc. Infiltrated leaves were harvested 48 h after inoculation, decoloured in ethanol 96% (v ⁄ v), washed in ethanol 50% (v ⁄ v), rinsed in 150 mM K2HPO4 (pH 9Æ5), and stained for 1 h in 150 mM K2HPO4 (pH 9Æ5) containing aniline blue 0Æ01% (w ⁄ v) (Sigma). Samples were mounted on 50% glycerol and examined using a fluorescence microscope (Olympus System BXS1) under UV (exc 385 nm). Callose deposits can be identified as bright spots on the leaf.

Quantitative reverse-transcriptase PCR (qRT-PCR) To quantify expression level of the PR-1 and PDF1Æ2 genes, qRT-PCR was performed. Total RNA was extracted with Trizol reagent (Invitrogen) from lemon leaves agroinfiltrated with the Bs2 gene expression construct (pLS-Bs2) or the negative control plasmid (pLS) and inoculated with wildtype Xcc or the avrBs2 mutant of Xcc at 24 h after agroinfiltration. Three plants per treatment were used and the experiment was carried out five times. The samples were taken at 0, 24, 48 and 72 h after the inoculation. All RNA was treated with DNase I to avoid DNA contamination and its quality was assessed by agarose gel electrophoresis and spectrophotometry. Specific primers for PDF1Æ2 and PR-1 genes were designed by using PRIMER3 software. Primer sequences were: PR1-f (5¢-GAC CGA TGA GAT GGG ACA AC-3¢) and PR1-r (5¢-GTA AGG CCG TTT ACC AGC AA-3¢) for PR-1 amplification and PDF1Æ2-f (5¢-GCT TCC ATC ATC ACC CTT ATC TTA TCT TC-3¢) and PDF1Æ2-r (5¢GGC TTC TCG CAC AAC TTC TG-3¢) for PDF1Æ2 amplification. In order to normalize the expression levels of the target genes, amplification of the actin gene was used, and the primer sequences were: ACT1 (5¢-TTT ACC ACC ACA GCC GAA CG-3¢) and ACT2 (5¢-TGG AGC CAC GAC CTT GAT-3¢). Production and amplification of cDNA was performed in one step by using the iScript One Step RT-PCR Kit SYBR Green (Bio-Rad) according to manufacturer’s instructions. Synthesis of cDNA was carried out at 50C for 10 min and RT inactivation at 95C for 5 min. PCR reactions were performed for 40 cycles of denaturation at 95C for 10 s and primer annealing and extension at 55C for 30 s. The reactions were carried out in triplicate and monitored in real time with the Miniopticon Real Time PCR Detector


Upgrade, for MJ mini from Bio-Rad. After amplification, melting-curve analysis excluded artefactual amplifications. The relative expression of RNA transcripts was quantified with the threshold cycle values (Ct) obtained from each sample by using the 2)DDCt method (Livak & Schmittgen, 2001). cDNA from leaves treated with the negative control plasmid was used as reference. The relative gene expression level is given by 2)DDCt, where – DD Ct can be calculated by using the following formula: D Ct = Ct

(PR-1 or PDF1Æ2)

(sample) ) D Ct (reference).

– Ct

(actin)

and DD Ct = D Ct

Changes in gene expression were reported as foldchanges in the PR-1 or PDF1Æ2 expression on lemon leaves treated with the pLS-Bs2 (sample), compared to expression in leaves treated with pLS (reference) at each time point after inoculation.

Statistical analysis All the experiments were performed in triplicate and results were subjected to ANOVA analysis and Student’s ttest by using the INFOSTAT 2008 software.

Results

(a)

Control

10

8

6

4

2

0 Control

Functional activity of the Bs2 gene in agroinfiltrated lemon leaves To check if the Bs2 resistance gene from pepper has functional activity in C. limon tissue, transient co-expression of the pepper-Bs2 and Xcv-avrBs2 genes in attached leaves was evaluated, to see if it generated a reduction in citrus canker disease. For this purpose, A. tumefaciens was transformed with different plasmid constructions: (a) the pLS-Bs2 plasmid carrying the Bs2 gene, (b) the 35S-avrBs2 plasmid containing the avrBs2 gene, or (c) the pLS plasmid without any of these genes as a negative control (Fig. 1). Agrobacterium tumefaciens containing pLS-Bs2 was mixed with an equal volume of cells containing the 35S-avrBs2 plasmid. This mixture of cells was handinfiltrated into intercellular leaf spaces of lemon leaves. As a control, A. tumefaciens containing only the pLS plasmid was infiltrated in comparable areas of the same leaf. Twenty-four hours after treatment, agroinfiltrated zones of leaves were wounded on the abaxial side and inoculated by spraying a suspension of Xcc (108 CFU mL)1). The methodological approach used for Xcc inoculation in this assay ensures the entry of the bacterium in the leaf. At 15 days after inoculation (dai), the development of cankers in the leaf area co-infiltrated with Bs2 and avrBs2 genes was reduced compared to the area infiltrated with the negative control (Fig. 2a). This observation was coincident with bacterial growth experiments where the CFU cm)2 leaf in co-infiltrated leaf zones was 1000-fold lower than the number recovered from negative control infiltration areas (Fig. 2b).

Bs2 + avrBs2

(b) 12

Log CFU cm–2 inoculated leaf

652

Bs2+avrBs2

Figure 2 Effect of the expression of Bs2 gene from pepper and avrBs2 from Xcv on citrus canker symptoms. Agroinfiltrations into lemon leaves were carried out as follows: one zone was agroinfiltrated with control construction (pLS) and a second zone with a mixture of an equal volume of Agrobacterium suspensions containing the pLS-Bs2 and the 35S-avrBs2 plasmid. Xcc inoculation was performed 24 h after agroinfiltration by nicking followed by spraying. Photos of disease symptoms were taken (a) and the Xcc population was determined (b) 15 dai by homogenizing the tissue in 10 mM MgCl2, followed by dilution plating. Note the reduced canker symptoms following Bs2 and avrBs2 co-expression. Values are expressed as means ± SD of three separate experiments.

These results indicate that lemon harbours the necessary cell machinery for functional activity of the Bs2 resistance gene from pepper.

The pepper Bs2 gene recognizes Xcc-AvrBs2 in lemon leaves leading to increased resistance To test the hypothesis that the Bs2 gene could recognize the avrBs2 of Xcc, the effect of Bs2 gene expression on citrus canker symptom development was evaluated in lemon leaves. To study this, lemon leaves were agroinfiltrated with pLS-Bs2 or pLS 24 h prior to inoculation either by spraying a suspension of Xcc (108 CFU mL)1) on the abaxial side of leaves wounded with a razor blade, or through syringe pressure infiltration of an Xcc suspension (105 CFU mL)1). A clear reduction of disease symptoms was observed 15 dai in areas of leaves pre-treated with the Bs2 gene construction, independent Plant Pathology (2012) 61, 648–657


(a)

(b)

653

(c)

Bs2

10 8 6 4 2 0

Control

Bs2

35 30 25 20 15 10 5 0

Control

Bs2

Canker number cm–2 of leaf

12

Canker number cm–2 of leaf

Log CFU cm–2 inoculated leaf

Control

30 25 20 15 10 5 0

Control

Bs2

Figure 3 Expression of Bs2 gene from pepper reduces bacterial growth and symptom production in Citrus limon leaves in response to Xcc. Citrus canker symptoms in lemon leaves infiltrated with Agrobacterium culture containing Bs2 construction (pLS-Bs2) or negative control construction (pLS) and inoculated with Xcc 24 h after treatment. Xcc inoculation was carried out using three methods: nicking followed by spraying (a), pressure infiltration (b), or cotton swab application of a bacterial suspension (c). Symptom quantification in the nick and spray method was determined by homogenizing the tissue in 10 mM MgCl2 followed by dilution plating, and expressed in CFU cm)2 of inoculated leaf; in the other two inoculation methods, average number of cankers developed per cm2 of inoculated leaf was determined. Values are expressed as means of 30 young leaves selected from three lemon plants. The experiment was carried out in triplicate. Photos of disease symptoms were taken 15 dai.

of the inoculation method used (Fig. 3). Leaves inoculated by nicking and spraying showed a reduction of bacterial growth by about one order of magnitude (Fig. 3a), whereas in leaves inoculated with pressure infiltration the average number of cankers per cm2 of inoculated leaf decreased from 33 to 22 in areas treated with pLS and pLS-Bs2, respectively (Fig. 3b). These results indicate that the presence of Bs2 inhibits development of disease symptoms associated with inoculation of high densities of Xcc. To mimic the natural infection of Xcc entering only through leaf stomata, thereby ruling out any interference caused by the wound or infiltration method, a similar experiment was conducted. Ten lemon leaves per plant were agroinfiltrated with the pLS-Bs2 and 10 leaves of other lemon plants with the negative control plasmid pLS, 24 h prior to inoculation with Xcc. Xcc was inoculated by applying a suspension (108 CFU mL)1) with a cotton swab on the abaxial side of agroinfiltrated leaves. In this experiment three plants were used for each treatment. The number of cankers developing on leaves treated with pLS-Bs2 was significantly less (P < 0Æ05) 15 dai, Plant Pathology (2012) 61, 648–657

than on those treated with the control plasmid pLS (Fig. 3c). A noticeable effect of this treatment was that inhibition of canker was observed, not only within but also beyond the agroinfiltrated area of the leaf. In order to determine if the reduced disease symptoms seen in leaves infiltrated with the Bs2 gene is a direct consequence of recognition of the Xcc-avrBs2 gene, a strain avrBs2 mutant of Xcc was generated by disruption of this gene (Fig. S1). At 24 h after Bs2 agroinfiltration (pLS-Bs2), a suspension of the mutant strain (105 CFU mL)1) was infiltrated. As expected, citrus canker symptoms did not decrease in Bs2 agroinfiltrated leaves relative to negative control treated leaves, unlike when wildtype Xcc was inoculated (Fig. 4). Further evidence of such an interaction was shown by inoculation of leaves, infiltrated previously with the Bs2 gene expression vector, with the avrBs2 mutant of Xcc complemented with the Xcc avrBs2 gene, where leaves showed a reduction of symptoms (data not shown). In a similar way, when the Bs2 gene was co-infiltrated with the Xvc-avrBs2 gene, disease symptom reduction was observed when plants were challenged with the avrBs2

654


Wildtype Xcc

(a)

Canker number cm–2 leaf

Canker number cm–2 leaf

30

30 25 20 15 10 5 0 Control wt Xcc

avrBs2 mutant of Xcc

(b)

35

Bs2

Bs2+avrBs2

25 20 15 10 5 0 Control Xcc mutant

Bs2

Bs2+avrBs2

Figure 4 Canker development in lemon leaves infected with wildtype Xcc or avrBs2 mutant of Xcc and previously agroinfiltrated with control, Bs2 and Bs2+ avrBs2 constructions. Lemon leaves were agroinfiltrated with negative control, Bs2 or a mixture of Bs2 and avrBs2 constructions. At 24 h after agroinfiltration, a suspension (105 CFU mL)1) of either wildtype Xcc (a) or avrBs2 mutant of Xcc (b) was infiltrated. The average number of cankers developed per cm2 of leaf was calculated and photos were taken 15 dai.

mutant of Xcc, as well as in plants inoculated with wildtype Xcc (Fig. 4). These data taken together suggest an interaction between the pepper Bs2 protein and the Xcc-avrBs2 gene product generating decreased canker disease symptoms in agroinfiltrated lemon leaves. Interestingly, no HR-associated necrosis could be observed in lemon leaves infiltrated with the pLS-Bs2 expression vector and Xcc or the co-infiltration of the pLS-Bs2 and the 35S-avrBs2 plasmid. To determine whether this phenomenon was due to a lemon-specific reaction, transient expression assays were performed in pepper plants susceptible to Xcv. In this assay, the A. tumefaciens strain containing the Bs2 construction was infiltrated into pepper leaves and inoculated 24 h later with a bacterial suspension of wildtype Xcc or avrBs2 mutant of Xcc. No HR reaction was observed in the leaves inoculated with wildtype Xcc or with the mutant. As positive control, pLS-Bs2 and Xcv-avrBs2 were co-infiltrated and HR was observed at 48 h after agroinfiltration, as described by Tai et al. (1999). However, HR was not observed in the leaf area agroinfiltrated with the XcvavrBs2 genetic construct or with the Bs2 construction alone (data no shown).

Bs2 gene agroinfiltration induces plant defence markers in the presence of Xcc To elucidate if the effect of the Bs2 gene infiltration is associated with the induction of plant defence mechanisms, cellular and genetic markers of plant defences were studied. Many studies have demonstrated that callose, a ß-(1,3)-glucan with (1,6) modifications, is required for disease resistance against some plant pathogens (Hamiduzzaman et al., 2005; Enrique et al., 2011). In order to test for callose deposition as a response to Bs2 gene expression and Xcc infection, lemon leaves pre-treated with either pLS or pLS-Bs2 constructions 24 h before being inoculated with wildtype Xcc or with the avrBs2

mutant of Xcc, were assessed by aniline blue staining. Cytological observations were performed at the site of treatment 48 h after bacterial inoculations, using ultraviolet fluorescence microscopy. Leaves pre-treated with the Bs2 construct showed a greater number of bright spots corresponding to callose deposits when challenged with the wildtype Xcc compared to leaves inoculated with the avrBs2 mutant of Xcc, or pre-treated with the plasmid control (Fig. 5). Transcript levels of the two pathogen-related genes PR-1 (salicylic acid pathway) and PDF1Æ2 (jasmonic acid Bs2

Control

Wildtype Xcc

avrBs2 mutant of Xcc Figure 5 Effect of Bs2 expression on callose deposition in lemon leaves inoculated with Xcc. Lemon leaves agroinfiltrated with negative control (pLS) or Bs2 (pLS-Bs2) constructions and inoculated either with wildtype Xcc or avrBs2 mutant of Xcc, were collected 2 dai, stained with aniline blue and analysed by fluorescence microscopy (UV). Micrographs were taken under UV excitation revealing aniline blue-stained callose deposits as white dots in these pictures. Arrows indicate callose deposits. The experiment was carried out in triplicate with similar results. Magnification 10·.



Relative expression of PR-1 and PDF1.2

6

PR-1 PDF1.2

5 4 3 2 1 0

0h

24 h

48 h

72 h

Time (h) Figure 6 qRT-PCR analysis of PR-1 and PDF1Æ2 expression. Average cycle threshold (Ct) values and n-fold changes in expression of the target genes (PR-1 and PDF1Æ2) were calculated in Bs2-expressing lemon leaves (sample), compared to Bs2-nonexpressing ones (reference) at different time points after wildtype Xcc inoculation. The Ct values were normalized against the internal control gene (b-actin) expression. The mean fold change in expression of the target genes at each time point was calculated using the 2)DDCt method (see Material and methods). The average values were calculated from experiments carried out five times.

pathway), both genetic markers of plant defence responses, were analysed by qRT-PCR. The comparative CT quantification (DDCt method) was applied to detect changes in PR-1 and PDF1Æ2 gene expression in leaves pre-treated with the Bs2 gene construction, compared with those pre-treated with the negative control and then inoculated with wildtype Xcc or the avrBs2 mutant. Relative quantification was performed using the b-actin gene as an endogenous expression control. As shown in Figure 6, PR-1 mRNA levels increased at least five-fold 24 h after wildtype Xcc inoculation in leaves pre-treated with the Bs2 construction, returning to initial levels of expression (at 0 h) after 72 h. In contrast, no significant changes in PDF1Æ2 mRNA levels were observed. However, there were no differences in expression levels of either gene when the avrBs2 mutant was inoculated (data not shown). The clear reduction in canker disease symptoms, and the lower bacterial growth combined with the induction of PR-1 and alterations in the plant cell wall, suggest that the interaction between Bs2 and Xcc is correlated with the induction of a plant defence response.

Discussion Genetic transformation is a promising alternative to generate new citrus genotypes, which could help solve many classical breeding problems. However, before initiating the time consuming process of transgenesis, it is recommended that the potential utility of a candidate gene is evaluated by transient gene expression studies if possible. This is even more important when working with woody Plant Pathology (2012) 61, 648–657

655

species that are problematic to regenerate in vitro, such as C. limon, and difficult to genetically transform (Ghorbel et al., 2000). As a consequence, transient gene expression experiments can be very useful, allowing rapid studies on the functional activity of a gene product of interest. Genetic resistance appears to be the most efficient form of protection against plant pathogens. By transforming susceptible plants with cloned R-genes, novel resistant lines can be rapidly generated. The R-genes usually confer race-specific resistance, and as such, their effectiveness and durability are based on their interaction with complementary pathogen avirulence genes (Thilmony et al., 1995). In general, when the disease resistance is controlled by a single R-gene, it may have limited durability in the field due to high mutation selection pressure. However, some genes, including Xa21 from rice, Bs2 from pepper and mlo from barley, have proved to be exceptionally durable in their natural host species. Moreover, they recognize multiple races of specific pathogens (Shah, 1997). Xa21 and Bs2 are thought to be durable because they recognize conserved components required for pathogen virulence or fitness (Dempsey et al., 1998). In the case of the pepper resistance Bs2 gene, the corresponding avrBs2 gene of Xcv shows a high degree of conservation with the avrBs2 genes isolated from Xanthomonas infecting other plant species (Kearney & Staskawicz, 1990). When analysing the protein database using the BLASTP program, it was observed that the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xcc (data not shown). Therefore, the hypothesis of this work was that the transfer of the Bs2 R-gene from pepper into any citrus species may confer resistance to citrus canker disease. Transient expression assays were used through agroinfiltration of a Bs2 gene expression construct in leaves of lemon and subsequent challenge with a pathogenic Xcc strain to evaluate the hypothesis above. The results showed direct evidence that the presence of Bs2 in lemon leaves produces a clear reduction in the severity of canker disease symptoms. Further proof of such an effect was evident when no decrease in disease symptoms was observed when an avrBs2 mutant of Xcc was used to infect plants after Bs2 gene infiltration, but the effect was recovered when the mutant was complemented with a wildtype avrBs2 gene. The presence of the Bs2 gene could be associated with a direct effect on bacterial growth and plant defence activation because an increase in callose deposition and induction of PR-1 gene expression were observed. All these results taken together suggest that the apparent decrease in disease symptoms observed by pretreatment with the Bs2 gene stems from an interaction with the avrBs2 avirulence gene of Xcc. Because of the high identity found between the AvrBs2 protein from Xcc and Xcv, it can be expected to have a similar functional response when interacting with the Bs2 gene product. However, co-infiltration of the avrBs2 gene from Xcv together with the resistance gene Bs2 produced a higher degree of disease symptom reduction in comparison to leaves agroinfiltrated with only the Bs2 gene. This result could be explained by taking into account the

656


different strategies to express the avrBs2 gene in the host. While the Xcv avrBs2 is co-transiently expressed with the Bs2 gene in the plant cell, both genes mediated by Agrobacterium, the Xcc AvrBs2 product must be delivered from the bacteria through a type III secretion system before interacting with the plant cell. Tai et al. (1999) reported that, due to the phenomenon referred to as restricted taxonomic functionality (RTF), the functional expression of the pepper R gene Bs2 in stable transgenic plants supports its use as a source of resistance only in other solanaceous plant species. They reached this conclusion based on the observation of absence of HR on non-solanaceous species by transient co-expression assay of Bs2 and Xcv-avrBs2 genes, but without testing a subsequent challenge against virulent pathogens. Although HR is a hallmark of gene-for-gene disease resistance, the relative importance of cell death in this form of disease resistance is not clear and may vary depending on the target pathogen species (Dangl et al., 1996). The literature provides some evidence that the HR is not always required for gene-for-gene resistance (Goulden & Baulcombe, 1993; Yu et al., 1998). The results observed in this study lead to the inference that the Bs2 gene could work in non-solanaceous species without HR development. In support of such a hypothesis it was observed that the level of protection in lemon was similar to that reported in transgenic tomato and pepper plants expressing the Bs2 gene (Tai et al., 1999). Important characteristics of the Bs2 gene in terms of resistance durability and ability to recognize avirulence genes from many different pathogenic strains of Xanthomonas, give this gene special importance in future molecular breeding strategies. According to the results presented, the Bs2 gene should be considered a promising candidate for stable transgenic expression into citrus species, thus contributing to the reduced use of environmentally detrimental disease management products such as copper.

Acknowledgements This project was partially supported by the EEAOC Citrus Program, PAV 137 ⁄ 6 and PICT 2006-02082 from ‘Agencia Nacional de Promocioń Cientı´fica y Tecnolo´gica’, and PIP 6441 from the Consejo Nacional de Investigaciones Cientı´ficas y Tećnicas (CONICET). MRM, AAV and APC are members of the CONICET. LNS, LR and RE are CONICET fellows. We thank Dr B. Staskawikz (University of California, Berkeley) for providing us with the Bs2 and avrBs2 genes, L. Montivero and A. Manes (EEAOC, Argentina) for reviewing the English version of the manuscript and Dr Welin (EEAOC, Argentina) for the critical reading of this manuscript.

References Barbosa-Mendes JM, Filho FdAAM, Filho AB, Harakava R, Beer SV, Mendes BMJ, 2009. Genetic transformation of Citrus

sinensis cv. Hamlin with hrpN gene from Erwinia amylovora and evaluation of the transgenic lines for resistance to citrus canker. Scientia Horticulturae 122, 109–15. Bespalhok Filho JC, Kobayashi AK, Pereira LFP, Vieira LGE, 2001. Transformacao de laranja visando resistencia ao cancro cı´trico usando genes de peptı´deos antibacterianos. Biotecnologia Cieˆncia Desenvolvimento 23, 62–6. Boscariol RL, Monteiro M, Takahashi EK et al., 2006. Attacin A gene from Tricloplusia ni reduces susceptibility to Xanthomonas axonopodis pv. citri in transgenic Citrus sinensis ‘Hamlin’. Journal of the American Society for Horticultural Science 131, 530–6. Brunings AM, Gabriel DW, 2003. Xanthomonas citri: breaking the surface. Molecular Plant Pathology 4, 141–57. Dangl JL, Dietrich RA, Richberg MH, 1996. Death don’t have no mercy: cell death programs in plant-microbe interactions. Plant Cell 8, 1793–807. Das AK, 2003. Citrus canker – A review. Journal of Applied Horticulture 5, 52–60. Dempsey DMA, Silva H, Klessig DF, 1998. Engineering disease and pest resistance in plants. Trends in Microbiology 6, 54–61. Enrique R, Siciliano F, Favaro MA et al., 2011. Novel demonstration of RNAi in citrus reveals importance of citrus callose synthase in defence against Xanthomonas citri subsp. citri. Plant Biotechnology Journal 9, 394–407. Federacioń Argentina Del Citrus, 2009. The Argentine Citrus Industry [http://www.federcitrus.org.ar/actividad-citricola2009.pdf]. Fehri LF, Sirand-Pugnet P, Gourgues G, Jan G, Wroblewski H, Blanchard A, 2005. Resistance to antimicrobial peptides and stress response in Mycoplasma pulmonis. Antimicrobial Agents and Chemotherapy 49, 4154–65. Flor HH, 1971. Current status of the gene-for-gene concept. Annual Review of Phytopathology 9, 275–96. Ghorbel R, Dominguez A, Navarro L, Penna L, 2000. High efficiency genetic transformation of sour orange (Citrus aurantium) and production of transgenic trees containing the coat protein gene of citrus tristeza virus. Tree Physiology 20, 1183–9. Goulden MG, Baulcombe DC, 1993. Functionally homologous host components recognize potato virus X in Gomphrena globosa and potato. Plant Cell 5, 921–30. Hamiduzzaman MM, Jakab G, Barnavon L, Neuhaus JM, MauchMani B, 2005. Beta-Aminobutyric acid-induced resistance against downy mildew in grapevine acts through the potentiation of callose formation and jasmonic acid signaling. Molecular Plant-Microbe Interactions 18, 819–29. Hammond-Kosack KE, Jones JDG, 1997. Plant disease resistance genes. Annual Review of Plant Physiology and Plant Molecular Biology 48, 575–607. Herrero R, Asıńs MJ, Pina JA, Carbonell EA, Navarro L, 1996. Genetic diversity in the orange subfamily Aurantioideae - II: genetic relationships among genera and species. Theoretical and Applied Genetics 93, 1327–34. Kearney B, Staskawicz BJ, 1990. Widespread distribution and fitness contribution of Xanthomonas campestris avirulence gene avrBs2. Nature 346, 385–6. Livak KJ, Schmittgen TD, 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(Delta Delta C(T)) Method. Methods 25, 402–8. Llave C, Kasschau KD, Carrington JC, 2000. Virus-encoded suppressor of posttranscriptional gene silencing targets a



maintenance step in the silencing pathway. Proceedings of the National Academy of Sciences, USA 97, 13401–6. Martıńez Zamora M, Castagnaro A, Dıáz Ricci J, 2008. Genetic diversity of Pto-like serine ⁄ threonine kinase disease resistance genes in cultivated and wild strawberries. Journal of Molecular Evolution 67, 211–21. Mendes BMJ, Cardoso SC, Boscariol-Camargo RL, Cruz RB, Mouraõ Filho FaA, Bergamin Filho A, 2010. Reduction in susceptibility to Xanthomonas axonopodis pv. citri in transgenic Citrus sinensis expressing the rice Xa21 gene. Plant Pathology 59, 68–75. Penã L, Navarro L, 1999. Transgenic citrus. In: Bajaj YPS, ed. Biotechnology in Agriculture and Forestry. Transgenic Trees. Berlin, Germany: Springer-Verlag 44, 39–53. Prentki P, Krisch HM, 1984. In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29, 303–13. Quandt J, Hynes MF, 1993. Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria. Gene 127, 15–21. Ronald PC, Albano B, Tabien R et al., 1992. Genetic and physical analysis of the rice bacterial blight disease resistance locus, Xa21. Molecular and General Genetics 236, 113–20. Shah DM, 1997. Genetic engineering for fungal and bacterial diseases. Current Opinion in Biotechnology 8, 208–14. Siciliano F, Torres P, Sendin L et al., 2006. Analysis of the molecular basis of Xanthomonas axonopodis pv. citri pathogenesis in Citrus limon. Electronic Journal of Biotechnology 9, 200–4. Stone BA, Clarke AE, 1992. The Chemistry and Biology of (1,3)-bGlucans. Bundoora, Victoria, Australia: La Trobe University Press. Swords KM, Dahlbeck D, Kearney B, Roy M, Staskawicz BJ, 1996. Spontaneous and induced mutations in a single open reading frame alter both virulence and avirulence in Xanthomonas campestris pv. vesicatoria avrBs2. Journal of Bacteriology 178, 4661–9. Tai H, Dahlbeck D, Clark E et al., 1999. Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato. Proceedings of the National Academy of Sciences, USA 96, 14153–8.


657

Thilmony RL, Chen Z, Bressan RA, Martin GB, 1995. Expression of the tomato Pto gene in tobacco enhances resistance to Pseudomonas syringae pv. tabaci expressing avrPto. Plant Cell 7, 1529–36. Yeaman MR, Yount NY, 2003. Mechanisms of antimicrobial peptide action and resistance. Pharmacological Reviews 55, 27–55. Yu IC, Parker J, Bent AF, 1998. Gene-for-gene disease resistance without the hypersensitive response in Arabidopsis dnd1 mutant. Proceedings of the National Academy of Sciences, USA 95, 7819–24.

Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. avrBs2 mutant of Xcc construction. (a) A 1259-bp fragment of chromosomal DNA containing a portion of the avrBs2 gene was amplified by PCR using Xcc chromosomal DNA as a template and cloned in pGEM T-Easy (Promega). The 2-kbp fragment containing a SpcR (X) cassette was ligated as a HindIII fragment into pGEM T-Easy digested with HindIII within the avrBs2 gene. The resulting construction was cloned as a 3259-bp EcoRI fragment into the sacB suicide vector pKmobsacB digested with EcoRI and later transferred by electroporation to Xcc. (b) Southern blot: Genomic DNA was prepared from the wildtype Xcc strain and the avrBs2 mutant. The DNA was digested with ClaI, separated on a 0Æ8% agarose gel and blotted. The blot was probed with the probe indicated in (a). By digestion with ClaI, DNA fragments of 844-bp and 2844-bp were expected for wildtype and mutant, respectively. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding authors for the article.