Inducible gene expression by Pepper huasteco virus in Capsicum chinense plants with resistance to geminivirus infections

Virology / Virologie

Inducible gene expression by Pepper huastecovirus in Capsicum chinense plants with resistanceto geminivirus infections

J.L. Anaya-López, E. Pérez-Mora, I. Torres-Pacheco, C.I. Muñoz-Sánchez, L. Guevara-Olvera, M. González-Chavira, N. Ochoa-Alejo, R.F. Rivera-Bustamante, and R.G. Guevara-González

Abstract: To understand the interaction between pepper (Capsicum chinense) and Pepper huasteco virus (PHV), geneexpression analyses using differential display reverse transcription – polymerase chain reaction (RT–PCR) were carriedout with plants (C. chinense accession BG-3821) previously identified as resistant to single and mixed geminivirusinfection. In vitro propagated virus-free C. chinense plants were used in our work to reduce variability among plants.With this strategy, 45 differentially expressed RT–PCR fragments were identified, and two of them were furtherconfirmed by Northern analyses as differentially induced when the plants were PHV infected. Sequencing of these RT–PCR fragments and comparison with GenBank showed some similarity with a bacterial methyl transferase gene (CbiL)and an NADP-malic enzyme of Sorghum bicolor. Aspects of the possible role of these genes in the interaction betweenC. chinense and PHV are discussed.

Key words: Capsicum chinense, differential display reverse transcription – polymerase chain reaction, geminivirus,Pepper huasteco virus, resistance.

Résumé : Afin de comprendre l’interaction entre le piment fort (Capsicum chinense) et le virus du piment huasteco(PHV), des analyses de l’expression génétique faisant appel à la technique transcription inverse – réaction en chaîne dela polymérase (TI–RCP) en différence de profil furent effectuées sur des plantes (C. chinense obtention BG-3821)préalablement identifiées comme résistantes à l’infection par un seul ou plusieurs géminivirus en mélange. DesC. chinense multipliés in vitro et exempts de virus furent utilisés dans nos travaux, de façon à réduire la variabilitéentre les plantes. Grâce à cette stratégie, 45 fragments TI–RCP exprimés par action différentielle furent identifiés, dontdeux furent, par la suite, confirmés par analyses de Northern comme induits par action différentielle lorsque les plantesfurent infectées par le PHV. Le séquençage de ces fragments TI–RCP et leur comparaison aux données de la GenBankont montré une certaine similitude avec un gène bactérien de la méhyltransférase (CbiL) et une enzyme NADP-maliquedu Sorghum bicolor. Des aspects du rôle possible de ces gènes dans l’interaction entre le C. chinense et le PHV sontdiscutés.

282Mots clés : Capsicum chinense, technique transcription inverse – réaction en chaîne de la polymérase en différence deprofil, géminivirus, virus du piment huasteco, résistance.

Can. J. Plant Pathol. 27: 276–282 (2005)

276

Accepted 12 January 2005.

J.L. Anaya-López and E. Pérez-Mora. Instituto Tecnológico de Celaya, Departamento de Ingeniería Bioquímica, Ave.Tecnológico y A. García-Cubas, S/N, Colonia FOVISSSTE, Apartado postal 57, Celaya, Guanajuato, Mexico, and InstitutoNacional de Investigaciones Forestales, Agrícolas y Pecuarias, Campo Experimental Bajío, Unidad de Biotecnología, CarreteraCelaya-San Miguel de Allende km 6.5, Apartado postal 112, Celaya, Gto, Mexico.I. Torres-Pacheco and M. González-Chavira. Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, CampoExperimental Bajío, Unidad de Biotecnología, Carretera Celaya-San Miguel de Allende km 6.5, Apartado postal 112, Celaya, Gto,Mexico.C.I. Muñoz-Sánchez, L. Guevara-Olvera, and R.G. Guevara-González.1 Instituto Tecnológico de Celaya, Departamento deIngeniería Bioquímica, Ave. Tecnológico y A. García-Cubas, S/N, Colonia FOVISSSTE, Apartado postal 57, Celaya, Guanajuato,Mexico.N. Ochoa-Alejo and R.F. Rivera-Bustamante. Centro de Investigación y de Estudios Avanzados del I. P. N-Unidad Irapuato,Carretera Irapuato-León, km 9.6, Libramiento Norte, Apartado postal 629, Irapuato, Gto, Mexico.

1Corresponding author (e-mail: [email protected]).

Anaya-López et al.: geminivirus / peppers / induced gene expressionIntroduction

The family Geminiviridae take their name from theunique structure of their geminate or twinned particle; theirgenomes consist of either one (monopartite) or two (bipar-tite) covalently closed circular single-stranded DNA (ssDNA)molecules (Hanley-Bowdoin et al. 1999). The familyGeminiviridae has been divided into four genera accordingto their host range, insect vector, and genome organization(Mastrevirus, Curtovirus, Begomovirus, and Topocuvirus)(Van Regenmortel et al. 2000). Pepper huasteco virus(PHV) is a member of the genus Begomovirus, has a bipar-tite genome (DNAs A and B), is transmitted by whiteflies,and naturally infects a series of dicotyledonous crops, in-cluding pepper (Garzón-Tiznado et al. 1993; Torres-Pacheco et al. 1996). In Mexico, PHV has been reported asone of the most important viral pathogens in pepper plants,and recently several sources of resistance in Mexican wildpeppers have been identified (Anaya-López et al. 2003;Godínez-Hernandez et al. 2001; Hernandez-Verdugo et al.2001). Among these sources of resistance to PHV are acces-sions of Capsicum chinense Jacq. collected from Yucatan,Mexico (accession BG-3821, Anaya-López et al. 2003),which are excellent candidates for studying the interactionbetween PHV and these resistant plants (Anaya-López et al.2003; Godínez-Hernandez et al. 2001). In previous work,we have shown that resistance to PHV in these plants mustbe due to restriction in viral movement within the plants,because PHV can replicate its genome in these plants, andwe observed a direct relationship between symptom levelsand the quantity of viral DNA (Godínez-Hernandez et al.2001). Thus, we have analyzed the differential gene expres-sion in these PHV-resistant C. chinense plants in the pres-ence and absence of the virus. The study of differentialgene expression could provide clues in the unraveling of theplant–pathogen interaction, and the differential display re-verse transcription – polymerase chain reaction (DDRT–PCR) technique has successfully been used in this way else-where (Benito et al. 1996; Kim et al. 2000; Muñoz-Sanchezand Bailey 1998). Our aim with this work was to initiate anunderstanding at the molecular level of resistance mecha-nisms in these plants to PHV infection, which could help tounderstand resistance to other geminiviruses and potentiallyother plant–pathogens interactions.

Materials and methods

Plant materialCapsicum chinense accession BG-3821 from the INIFAP

germplasm bank was used in this work. These plants werepreviously identified as resistant to single and mixedgeminivirus infections (Anaya-López et al. 2003; Godínez-Hernandez et al. 2001). Additionally, Capsicum annuum L.‘Sonora Anaheim’ was used as an experimental control forvirus infectivity.

In vitro propagation of C. chinense and evaluation ofvirus-free state of the plants

Capsicum chinense accession BG-3821 was propagatedin vitro using apical shoot meristem cultures as described inChristopher and Rajam (1994) and Madhud and Rajam

(1993). The virus-free state of these plants was determinedusing PCR with specific oligonucleotides designed to am-plify a 211-bp DNA fragment of the PHV component Acommon region. The specific primers used were 240 (5′-GGCTTATTTGTAATAAGAG-3′) and 241 (5′-GAATTA-AAGGTACATGGAC-3′) (Torres-Pacheco et al. 1996). Tocarry out PCR, DNA samples from upper and lower leavesof the in vitro propagated plants were obtained using a stan-dard protocol (Dellaporta et al. 1983).

Virus inoculationInoculation of PHV DNAs was carried out using a

biolistic procedure with a particle delivery system (modelPDS 1000, Dupont, Wilmington, Delaware). The PHV ge-nome used in this work was originally isolated and clonedfrom infected pepper plants collected in Tamaulipas, Mex-ico (Garzón-Tiznado et al. 1993). Dimeric clones of PHVcomponents A and B or bluescript SK + plasmid DNA(Stratagene, La Jolla, California) were used in particlebombardment. Bombardments used 5 µg of DNA from PHV(2.5 µg of each genomic components A and B); this inoculumwas divided up among six plants as described in Anaya-López et al. (2003) and Godínez-Hernandez et al. (2001).Plants were inoculated at the apical zone when they were atthe four-leaf stage, which is the optimal stage for infectingpepper with geminiviruses using the biolistic procedure(Godínez-Hernandez et al. 2001). Inoculated plants were in-cubated at 24 to 32 °C in a greenhouse. Test plants were in-oculated with dimeric PHV clones (components A and B),while control plants were mock-inoculated with bluescriptSK + plasmid DNA.

Differential display analysesThree days postinoculation plants were verified for PHV

infection by specific PCR analysis as mentioned above.Once it was determined whether the plants were infectedwith PHV, total RNA extraction of newly developed (upper)leaves from mock- and PHV-inoculated plants was carriedout by using a TRIzol commercial kit (Invitrogen, Carlsbad,California). Differential display (Liang and Pardee 1992)was carried out using a commercial kit (Delta differentialdisplay kit, ClonTech, Palo Alto, California). Two randomand two anchored primers, chosen randomly, were used inthese experiments with their respective P–T primer possiblecombinations. The sequences of the primers were as fol-lows: P1, 5′-ATTAACCCTCACTAAATGCTGGGGA-3′; P7,5′-ATTAACCCTCACTAAATGCTGTATG-3′; T6, 5′-CAT-TATGCTGAGTGATATCTTTTTTTTTCG-3′; T3, 5′-CAT-TATGCTGAGTGATATCTTTTTTTTTAG-3′. Advantage KlenTaq DNA polymerase (ClonTech, Palo Alto, California) wasused as recommended by the manufacturer. Display of DNAfragments obtained was carried out on 7 mol/L urea – 6%polyacrylamide gels in a DNA sequencing system (E-C Ap-paratus, New Haven, Connecticut) and visualized by silverstaining (Silver Sequence staining reagents, Promega, Madi-son, Wisconsin).

Cloning and sequencing DNA fragmentsDifferential amplified DNA fragments were ligated in PCR

2.1 TOPO Vector, and TOP 10 (F-mcrA∆(mrr-hsdRMS-mcrBC)φ80lacZ∆M15∆lacX74recA1araD139D(ara-leu)7697

Anaya-López et al.: geminivirus / peppers / induced gene expression 277

galUgalKrpsL(StrR)endA1nupG) one-shot chemically com-petent E. coli cells (Invitrogen, Carlsbad, California) wereused for transformation.

Northern blotThe Northern analysis was accomplished as described by

Sambrook et al. (1989). RNA was extracted using theTRIzol system (Invitrogen, Carlsbad, California) according tomanufacturer. Following electrophoresis in denaturing agarosegels, samples of total RNA from tested and control plantswere transferred onto Hybond N membranes (Amersham In-ternational plc; Little Chalfont, UK) for hybridization withJLC 220 RT–PCR fragment labeled with 32P-dCTP by ran-dom priming. The exposed X-ray film was scanned, and thehybridization signals were quantified with a digital imagesystem (1D Image Analysis Software Version 3.0.2, Kodakdigital system, Rochester, New York).

Sequencing and BLAST comparisonNucleotide sequences of the RT–PCR fragments analyzed

and generated by differential display were obtained bydideoxynucleotide chain termination method using an auto-matic sequencer (Applied Biosystems, Foster City, Califor-nia). DNA sequences of JLC 220, JLC 250, and JLC 280clones were deposited in GenBank with accession Nos.AY344114, AY819663, and AY345126, respectively. DNAsequences were analyzed by using the BLASTX andBLASTP algorithms from the National Center for Biotech-nology Information.

Results

PHV infectivity in virus-free C. chinense BG-3821Capsicum chinense BG-3821 was propagated in vitro,

and the plants determined to be virus-free by PCR wereused for virus inoculation by particle bombardment (Fig. 1).In the bombardment experiments, tested C. chinense plantswere inoculated with dimeric clones of PHV components Aand B or mock inoculated. As an additional control to eval-uate the infectivity of the PHV dimeric clones, a widelyused susceptible host for PHV (C. annuum ‘Sonora Ana-heim’) was inoculated (Anaya-López et al. 2003; Godínez-Hernández et al. 2001). Capsicum annuum ‘Sonora Ana-heim’ plants displayed typical PHV symptoms 5 dayspostinoculation. In contrast, C. chinense plants did not dis-play symptoms throughout the 30-day investigation period.At 3 days postinoculation, C. chinense plants were evalu-ated for the presence of PHV by PCR of DNA extractionsfrom upper and lower leaves with respect to the four-leafstage in which the plants were inoculated. This analysisshowed that PHV infected only the tested plants (notshown); thus, we could continue with the differential geneexpression study of these PHV-infected C. chinense plants.

Differential gene expressionTo evaluate differential gene expression in C. chinense

BG-3821 in infected and uninfected individuals, total RNAextractions from the same leaves as those sampled in thePCR analyses of these plants mentioned above were carriedout. Aliquots of 2 µg of total RNA were used to develop thedifferential display technique. A typical display of silver-

stained RT–PCR fragments with the P–T primer combina-tions used was obtained (Fig. 2A). To accept a RT–PCRfragment as differentially expressed in our system, two ar-bitrary categories were considered: (i) overexpressed frag-ments and (ii) fragments completely induced or repressedby PHV infection. Figure 2 shows an example of a differen-tially expressed DNA fragment in PHV-infected plants(fragment JLC 220) in two samples (Fig. 2B, lanes 3 and4). With this strategy, 45 differentially expressed RT–PCRfragments were identified; further, some of these were se-quenced, and two were analyzed by Northern blot. The RT–PCR fragments differentially expressed were obtained withthe different P–T primer combinations, and the fragmentsranged in size from 180 to 400 bp. Of these fragments, 9were completely induced, 6 completely repressed, and 30overexpressed by PHV infection (Table 1).

Northern blot and sequence analysesThree RT–PCR fragments with sizes ranging from ap-

proximately 200 to 400 bp, of which two were completelyinduced (category 2) and one was overexpressed (category1) only when C. chinense BG-3821 was PHV infected, wereselected for further characterization by sequencing andNorthern blot. These RT–PCR fragments were named JLC220, JLC 250, and JLC 280 based on their apparent size,and their sequences were obtained (Table 1 and Fig. 3).Fragments JLC 220 and JLC 280 were completely induced,and JLC 250 was overexpressed (Table 1). Sequence com-parison of fragment JLC 280 at protein level using the Na-tional Center for Biotechnology Information algorithmsshowed an identity of 36% with a NADP-malic enzyme ofSorghum bicolor (L.) Moench (Table 1 and Fig. 4). With thesame analysis, fragment JLC 250 showed an identity of

278 Can. J. Plant Pathol. Vol. 27, 2005

Fig. 1. Detection of Pepper huasteco virus (PHV) within invitro propagated Capsicum chinense plants by specificpolymerase chain reaction analysis. Lane 1, molecular weightmarker 1 kb; lane 2, positive control (a PHV-infected Capsicumannuum ‘Sonora Anaheim’); lane 3, negative control (a mock-inoculated C. chinense BG-3821); lanes 4–9, six differentC. chinense BG-3821 plants produced by in vitro apical shootmeristem cultures.

80% with a hypothetical protein of Neurospora crassa L.(Table 1 and Fig. 4). On the other hand, the putative trans-lated protein of JLC 220 showed some identity (51%) withCbiL protein (a precorrin-2 C20 methyltransferase) of Pro-pionibacterium freudenreichii van Niel (Fig. 4). Methyl-transferase activities have been associated with resistance inplants to some viruses (Vaucheret et al. 2001); meanwhile,it has been reported that NADP-malic enzyme expression isinduced when Cucurbita pepo L. cotyledons are infectedwith Cucumber mosaic virus (CMV) (Havelda and Maule2000; Tecsi et al. 1996). Thus, to study in more detail theexpression of JLC 220 and JLC 280 fragments, Northernblot analyses were carried out with these fragments asprobes, and the results showed that expression of both JLC220 and JLC 280 was only detected when C. chinense BG-

3821 was PHV infected (Fig. 5). These latter results indi-cate the specific induction of expression of JLC 220 andJLC 280 RT–PCR fragments by PHV infection in C. chi-nense BG-3821. Additionally, the Northern analysis wasalso carried out using as a probe the JLC 220 RT–PCRfragment on RNAs from PHV-susceptible plants ofC. annuum ‘Sonora Anaheim’ when PHV infected, and thesignal was not detected (Fig. 5).

Discussion

The study detected the differential expression of severalRT–PCR fragments in C. chinense BG-3821 when infectedby PHV. Differential expression of JLC 220 and JLC 280was confirmed by Northern analysis. It is worth mentioning

Anaya-López et al.: geminivirus / peppers / induced gene expression 279

Fig. 2. A representative result in differential display reverse transcription – polymerase chain reaction assays. (A) A section ofdenatured 6% polyacrylamide gels showing some displayed DNA fragments using P7 and T3 primers (“control” is the internal controlof the differential display kit used). (B) An enlarged section of (A), showing an example of a differentially expressed DNA fragmentin Pepper huasteco virus (PHV) infected plants (fragment JLC 220). Lanes 1 and 2, samples from Capsicum chinense BG-3821 plantsinoculated only with plasmid bluescript (pBS); lanes 3 and 4, samples from PHV-inoculated C. chinense BG-3821 plants.

Clone Primer set Size (bp) Nature of expression Similar protein (I, P (%))*

JLC 220 P7–T3 220 Completely induced CbiL (51, 75)JLC 250 P1–T6 255 Overexpressed Hypothetical protein of Neurospora crassa (80, 90)JLC 280 P7–T6 280 Completely induced NADP-malic enzyme (36, 58)

Note: A total of 45 gene fragments ranging in size from 180 to 400 bp were identified; of these, 9 were completely induced, 6were completely repressed, and 30 were overexpressed by PHV infection. Data in the table are only for those clones sequenced; theother 42 clones have not yet been sequenced.

*According to comparison with GenBank. I, percent identity; P, percent identity when positives or similar amino acids wereconsidered.

Table 1. Data from the three gene fragments differentially expressed in the study.

that in our study in vitro propagated plants were used. The-oretically, this represents a cleaner strategy in comparisonto other similar studies carried out in pepper, in which dif-ferential gene expression in response to a virus infectionwas detected (Lee et al. 2001; Park et al. 2002). The se-quence comparisons with GenBank displayed an identity of51% with a C-terminal segment of CbiL protein ofP. freudenreichii subsp. shermanii; this identity was 75%when considering similar amino acids in this segment. CbiL

is a precorrin C20 methyltransferase involved in cobalaminand siroheme biosynthesis in P. freudenreichii (Roessner etal. 2002). Methyltransferases constitute a protein familythat in some cases shows no substantial homology amongits members, but they all catalyze the transfer of methylgroups to several substrates including amino acids andDNA (Schubert et al. 2003). Although speculative, this re-sult suggests a possible role for the protein encoded by thecomplete gene of JLC 220 in DNA methylation or maybe

280 Can. J. Plant Pathol. Vol. 27, 2005

Fig. 3. DNA sequence of JLC 220, JLC 250, and JLC 280 fragments, detected by differential display reverse transcription –polymerase chain reaction.

Fig. 4. Alignment between putative proteins of JLC 220, JLC 250, and JLC 280 and a CbiL protein, a hypothetical protein ofNeurospora crassa, and a NADP-malic enzyme, respectively. “+” indicates chemically similar amino acids. The alignment was carriedout using BLASTP algorithm of the National Center for Biotechnology Information.

in other types of stress response in plants to a pathogen.DNA methylation has been mentioned elsewhere as impor-tant in maintenance of methylation patterns and post-transcriptional gene silencing in plants (Cao and Jacobsen2002; Paszkowsky and Whitman 2001). Interestingly, allArabidopsis thaliana mutants in post-transcriptional genesilencing are hypersusceptible to CMV infection, suggest-ing that this phenomenon is necessary in a resistance mech-anism in this plant to a virus (Vaucheret et al. 2001). Inaddition, precorrin C20 methyltransferase and uroporphyrinIII C-methyltransferase are important enzymes in siroheme

and cobalamin biosynthesis. As well, siroheme is an impor-tant prosthetic group for enzymes as sulfite and nitritereductase, and it is considered as important in nitrate and sul-fate assimilation in plants (Leustek et al. 1997). Additionally,uroporphyrin III C-methyltransferase genes have been iso-lated from A. thaliana and maize (Leustek et al. 1997;Sakakibara et al. 1996). Sulfite reductase activity has beenrelated to sulfur uptake processes in plants, and this assimila-tion in higher plants is a crucial factor determining plant re-sistance to pests and pathogens (Hofgen et al. 2001). Thus,indirectly, the siroheme biosynthesis in C. chinense BG-3821 could be related to sulfur uptake and virus resistance.On the other hand, it has been reported elsewhere that CMVcan induce expression of NADP-malic enzyme in appar-ently uninfected cells of C. pepo cotyledons (Havelda andMaule 2000). This result could suggest that changes in ex-pression of NADP-malic enzyme by the host signal an im-minent infection; thus the plant is prepared for defenseagainst the pathogen. With our results, we cannot concludethat the role of JLC 220 and JLC 280 gene fragments ofC. chinense BG-3821 against PHV infection is located inpost-transcriptional gene silencing, efficiency of mineralsuptake, or as a signaling of infection, but these constituteintriguing hypotheses to be further evaluated. Additionally,it is worth mentioning that in our study, gene expression ofJLC 220 was not detected in a reportedly susceptible hostfor PHV (C. annuum ‘Sonora Anaheim’; Godínez-Hernández et al. 2001; Anaya-López et al. 2003). This re-sult could be due to low homology between the JLC 220probe with C. annuum RNA, or the encoded gene for JLC220 may be absent in C. annuum. Current efforts concen-trate on the isolation and characterization of the JLC 220and JLC 280 complete genes of C. chinense BG-3821 aswell as on the analysis of the rest of the DNA fragmentsdifferentially expressed in this study. Finally, the DDRT–PCR technique combined with silver staining to visualizethe DNA fragments expressed in our system was an effi-cient, simple, and reproducible method to precisely detectdifferential gene expression as mentioned elsewhere(Bockelman et al. 1999; Deng et al. 1999).

Acknowledgements

We acknowledge CONACYT (J31638-B, K325-P9702),CONCYTEG (01-09-201-088), CoSNET (567.01-P), and IFS(C/2955) for financial support of this research. J.L. Anaya-López and E. Pérez-Mora also acknowledge CONACYT,CoSNET, and CONCYTEG (02-24-203-004) for fellowshipsupport.

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Fig. 5. Northern blot analysis of Capsicum chinense BG-3821(A, B, and C) and Capsicum annuum ‘Sonora Anaheim’ (D andE) Pepper huasteco virus (PHV) inoculated or mock-inoculatedplants. Ten micrograms of total RNA was loaded in a denatured1.5% agarose gel, electrophoresed, and transferred to a Hybondplus membrane. Lane 1, RNA from PHV-inoculated plant; lane2, RNA from mock-inoculated plant. For (A) and (D) the probeis a JLC 220 reverse transcription – polymerase chain reaction(RT–PCR) fragment, for (B) the probe is a JLC 280 RT–PCRfragment, and for (C) and (E) the probe is a human 18 Sribosomal DNA.

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