Expression of hydroxymethylglutaryl-coa reductase 2 (HMG2) gene in chilli (Capsicum annuum L.) CM334 infected by Nacobbus aberrans and Phytophthora capsici

Expresión del gen HMG2 (hidroximetilglutaril-coa reductasa 2) en chile (Capsicum annuum L.) CM334 infectado por Nacobbus aberrans y Phytophthora capsici

Edgar Villar-Luna¹, Reyna I. Rojas-Martínez¹, Benito Reyes-Trejo², Mario Rocha-Sosa³, Emma Zavaleta-Mejía^1*

¹ Fitopatología. Campus Montecillo. Colegio de Postgraduados. 56230. Montecillo, Estado de México * Author for correspondence (zavaleta@colpos.mx).

³ Departamento de Biología Molecular de Plantas. Instituto de Biotecnología. Universidad Nacional Autónoma de México. Avenida Universidad 2001, Apartado Postal 510-3. 62210. Cuernavaca, Morelos.

Recibido: abril, 2014.
Aprobado: diciembre, 2014.

Abstract

Plants of chilli Capsicum annuum L. CM334 show a high level of resistance to Phytophthora capsici, but are susceptible to Nacobbus aberrans; such resistance has been associated with capsidiol accumulation, a sesquiterpene phytoalexin. In the present study the expression of hydroxymethylglutaryl-CoA reductase 2 gene (HMG2, associated with sesquiterpene phytoalexins biosynthesis) in CM334 chilli roots inoculated with N. aberrans and in combination with P. capsici, was determined by qRT-PCR. The levels of HMG2 transcripts were significantly reduced (p≤0.05) from -1.57 to -1.28-fold in the interaction CM334/N. aberrans; whereas in roots inoculated only with the oomycete, the accumulation of transcripts occurred earlier and at higher levels (1.52 to 3.54-fold). In plants inoculated with both pathogens, the expression was higher compared with those inoculated with the nematode only (p≤0.05), but generally lower than the expression observed in roots of plants inoculated with the oomycete alone. The expression of the HMG2 gene associated with defense was reduced in CM334 chilli plants infected by N. aberrans.

Key words: Capsicum annuum, capsidiol, real-time PCR, root-knot nematodes.

Plantas de chile Capsicum annuum L. CM334 exhiben un alto grado de resistencia a Phytophthora capsici, pero son susceptibles a Nacobbus aberrans; tal resistencia se ha asociado con la acumulación de capsidiol, una fitoalexina sesquiterpé-nica. En el presente estudio se determinó mediante qRT-PCR los niveles de expresión del gen HMG2 (hidroximetilglutaril-CoA reductasa 2, asociado con la biosíntesis de fitoalexinas sesquiterpénicas) en raíces de chile CM334 inoculadas con N. aberrans y en combinación con P. capsici. Los niveles de transcritos de HMG2 fueron significativamente reducidos (p≤0.05) de -1.57 a -1.28 veces en la interacción CM334/N. aberrans; mientras que en raíces inoculadas sólo con el oomiceto la acumulación de transcritos fue temprana e intensa (1.52 a 3.54 veces). En plantas inoculadas con ambos patógenos, la expresión fue alta comparada con aquellas inoculadas sólo con el nematodo (p≤0.05), pero generalmente menor que la expresión observada en raíces inoculadas sólo con el oomiceto. La expresión del gen HMG2 (asociado con defensa) fue reducida en raíces de chile CM334 infectadas por N. aberrans.

Palabras clave: Capsicum annuum, capsidiol, PCR en tiempo real, nematodos agalladores.

INTRODUCTION

The oomycete Phytophthora capsici Leonian limits the production of chilli pepper (Capsicum annuum L.) worldwide (Erwin and Ribeiro, 1996) in the field and greenhouse. The use of genotypes resistant to the oomycete is an economic and environmentally friendly strategy for disease management. The landrace Criollo de Morelos 334 (CM334) displays a high level of resistance to multiple isolates of P. capsici, which is expressed as a hypersensitive response in root, stem and foliage (Candela etal., 2000; Ueeda et al., 2006).

Plant defense mechanisms commonly involve the transcriptional activation of several defense-related genes; such activation leads to the de novo synthesis of a variety of proteins and antimicrobial compounds. In chilli plants, defense-related genes HMG2 (hydroxymethylglutaryl-CoA reductase), SC (sesquiterpene cyclase) and EAS (5-epiaristolochene synthase), which are involved in the biosynthesis of sesquiterpenic phytoalexins such as capsidiol, are over-expressed in response to attack by P. capsici (Ha et al., 2003; Silvar et al., 2008; Fernández-Herrera et al., 2012).

The hydroxymethylglutaryl-CoA reductase (HMGR) catalyzes the mevalonate synthesis, is encoded by a multigene family that in C. annuum includes HMG1, HMG2, and HMG3 (Ha et al., 2003). In plants, these genes are differentially regulated and lead to the biosynthesis of different types of isoprenoids; thus, in Arabidopsis thaliana, the expression of HMG1 and HMG2 is associated with sterols and triterpenes biosynthesis (Ohyama et al., 2007). Also, in Solanum tuberosum HMG1 is associated with sterol biosynthesis, whereas HMG2 and HMG3 are involved in sesquiterpene phytoalexins biosynthesis in response to attacks by pathogens (Choi et al., 1992).

Even though CM334 is highly resistant to the oomycete, the plants show susceptibility when they were previously infected by the nematode Nacobbus aberrans Thorne and Allen, 1944 (Trujillo-Viramontes et al., 2005). This phenomenon is associated with transcriptional and metabolic changes induced by the nematode in the root, including those pathways involved in defense (López-Martínez et al., 2011; Fernández-Herrera et al., 2012). In susceptible hosts, root-knot nematodes induce the formation of specialized feeding sites (SFS), a process that involves the down-regulation and up-regulation of genes. For instance, in the interaction Meloidogyne incognita/A. thaliana, genes involved in jasmonic acid/ethylene-dependent defense pathways and those involved in the synthesis of compounds with antimicrobial potential were down-regulated locally; in contrast, those related to the differentiation and maintenance of the SFS were up-regulated (Jammes et al., 2005). In CM334 chilli roots infected by N. aberrans and inoculated with P. capsici, there was a delay in EAS transcripts accumulation in comparison to those inoculated only with the oomycete; this event was related to a reduced accumulation of capsidiol (Fernández-Herrera et al., 2012). Based on this background, it is feasible to speculate that a reduction in the expression of defense-related genes could facilitate the establishment and successful reproduction of N. aberrans in CM334 plants, conditions that could be favorable for the infection by P. capsici. To contribute to the knowledge about the transcriptional changes induced during the complex interactions between the sedentary plant parasitic nematodes and their host plants, the aim of the present study was to determine the transcripts accumulation of the HMG2 gene in CM334 plants roots inoculated with N. aberrans alone and in combination with P. capsici.

Chilli plants and inoculation with N. aberrans and P. capsici

CM334 chilli plants were grown singly into pots containing 25 cm³ of sterile sand. The inoculum preparation, inoculation and time of inoculation were according to Fernandez-Herrera et al. (2012).

Assay establishment

Treatments were: 1) non-inoculated CM334 plants (control); 2) CM334 plants inoculated with P. capsici (Pc, 300 000 zoospores per plant); 3) CM334 plants inoculated with N. aberrans (N, 2000 second-stage juveniles, J₂); 4) CM334 plants inoculated with both pathogens (NPc); each treatment had 45 plants. At 6, 12 and 24 h after inoculation with the oomycete (haio), for each treatment and time the roots of 15 plants were frozen with liquid nitrogen and stored at -80 °C. The experiment was repeated once.

RNA extraction, cDNA synthesis and real-time PCR

Total RNA was extracted from frozen root tissues using the RNeasy^TM Plant mini kit and including a DNase treatment (Qiagen) following the manufacturer's instructions. The purity and integrity of RNA was verified by spectrophotometry (ND-1000, Nanodrop Technologies) (Thermo Scientific, USA) and by 1.2 % denaturing agarose gel electrophoresis, respectively. First-strand cDNA was synthesized from 2 µg of total RNA using the oligo(dT₁₅ primer (Promega) and M-MLV reverse transcriptase (Promega) according to manufacturer's instructions.

The expression levels of HMG2 gene (AF110383) were determined by real-time PCR in the ABI7500^TM system (Applied Biosystems) using the following primers: 5'-ATTACCTTCAGAATGAATACGCT-3' (forward) y 5'-CTCTCTATGTTTTGTGCTGGGT-3' (reverse). The reaction mixture consisted of buffer 10X, 1.5 mM MgCl₂, 0.4 µM of each primer, 0.2 mM dNTPs, amplificase (Biotecmol), SYBR^TM Green I (1: 75000) (Molecular Probes, Eugene, OR) as the reporter fluorophore, 10 nM fluorescein as passive reference, 2 µL cDNA, and nuclease-free water were added to a final volume of 25 µL. Amplification conditions consisted of an initial denaturation at 95 °C for 3 min, followed by 30 cycles at 95 °C for 15 s, annealing at 60 °C for 35 s, and extension at 72 °C for 35 s; the data were collected during the extension step. Dissociation curve analysis was performed to rule out amplification of non-specific products. Six technical replicates were performed for each treatment and each time. Glyceraldehyde-3-phosphate dehydrogenase gene (AJ246011) [5'-GGCCTTATGACTACAGTTCACTCC-3' (forward) y 5'-GATCAACCACAGAGACATCCACAG-3' (reverse)] was used as internal reference to normalize expression level, and control plants to calibrate expression levels of HMG2 gene, which was expressed as fold-change due to treatment in relation to the transcript basal levels in control plants (1x). Relative expression was calculated using the 2^-ΔΔCt method (Schmittgen and Livak, 2008). PCR products were purified with QIAquick^TM PCR purification Kit (Qiagen) according to the manufacturer's instructions and sequenced to confirm their identity.

An ANOVA was performed with the data (fold-change), the experimental design was completely randomized, and when significant differences were detected, the means of treatments were compared using Tukey's test (p≤0.05). These procedures were performed in SAS version 9.0 (SAS Institute Inc., 2002).

At all times, the HMG2 transcript levels were significantly reduced (p≤0.05) from -1.57 to -1.28-fold, in CM334 plant roots infected by N. aberrans alone (N treatment) and they were below the basal expression in control plants (Figure 1). In contrast, plant roots inoculated with P. capsici only (Pc treatment) showed the highest levels of expression (1.52 to 3.54-fold) (p≤0.05); in this incompatible interaction (CM334-P capsici), the maximum expression of the gene was observed at 6 haio (3.54-fold) and then decreased.

At 6 and 12 haio the transcript levels were reduced by 1.30 to 2.44-fold (p≤0.05) in plants inoculated with both pathogens (NPc treatment) as compared to those in plants of the Pc treatment (1.52 to 3.54-fold), but at 24 haio the reduction was not significant in comparison to the plants inoculated with the oomycete alone. In plants of the NPc treatment there was a delay in the expression of the gene, as the maximum expression of HMG2 was observed until the 12 haio (2.44-fold) and decreased at 24 haio (Figure 1).

The hydroxymethylglutaryl-CoA reductase (HMGR) catalyzes the first committed step of the mevalonate pathway for phytosterol and sesquiterpene biosynthesis; in plants, it is encoded by HMG genes, a multigene family that in C. annuum L. includes HMGI, HMG2, and HMG3 (Ha et al., 2003). These genes are differentially expressed during plant development and in response to pathogens or other stresses, for this particularity, HMGR is critical in the metabolic channeling of pathway intermediates towards specific end products, such as phytosterol and sesquiterpene metabolites, these last with antimicrobial properties (Weissenborn et al., 1995). In the present study, CM334 chilli plants of the Pc treatment, generally exhibited high levels of HMG2 transcripts. This result is consistent with that reported by Ha et al. (2003), who also found that increased expression of HMG2 was related with a strong expression of SC gene (sesquiterpene cyclase) in C. annuum cv. NocKwang inoculated with P. capsici; these researchers suggest that the coordinated regulation of both genes could be related with sesquiterpene phytoalexin accumulation in response to the infection. At 6 haio was evident that plants of the NPc treatment, had lower accumulation of HMG2 transcripts compared to Pc treatment, whereas in plants infected by the nematode alone, the levels recorded were lowest. This behavior was expected, since N. aberrans establishes a compatible interaction with chilli CM334. In several genotypes of chilli pepper, including CM334, the sesquiterpene phytoalexin capsidiol is associated with resistance to P. capsici (Candela et al., 2000), and capsidiol is also toxic to N. aberrans (Godínez-Vidal et al., 2010). These reports indicate that changes in gene-expression patterns of HMG2 might consequently result in a reduced accumulation of sesquiterpene phytoalexins such as capsidiol, which in turn could promote the establishment and successful reproduction of N. aberrans and P. capsici in chilli CM334.

Like Meloidogyne, Heterodera, and Globodera species, N. aberrans induces the formation of specialized feeding sites (SFS) to obtain essential nutrients for its development and reproduction (Manzanilla-López et al., 2002). Morphological changes in host root cells during the differentiation of these sites (giant cells or syncytia) are accompanied by drastic alteration in gene-expression patterns, inducing profound modifications in normal metabolism (Jammes et al., 2005). Down-regulation of genes involved in jasmonic acid/ethylene-dependent defense pathways, and also those involved in the synthesis of antimicrobial compounds, was reported in compatible plant-nematode interactions; in contrast, those related to the differentiation and maintenance of the SFS are up-regulated (Jammes et al., 2005). For instance, Heterodera glycines reduced the expression of OPR1 and OPR2 genes related to the jasmonic acid biosynthesis in a susceptible cultivar of Glycine max (Ithal et al, 2007). The down-regulation of defense genes that encode for pathogenesis-related proteins (PRs) and for WRKY transcription factors was reported by Jammes et al. (2005) in other compatible interactions, such as A. thaliana-M. incognita.

CONCLUSIONS

Nacobbus aberrans reduced the expression of HMG2 gene and such reduction was of greater magnitude when CM334 plants were infected only with the nematode in comparison to those inoculated with the two pathogens, N. aberrans and P. capsici or P. capsici.

ACKNOWLEDGMENTS

We thank the National Council on Science and Technology (CONACYT) for the scholarship granted to the first author and the financial support for the development of this investigation (Project 46331 Z-CONACYT) and Dr. Rosa Luisa Santillán-Baca for reviewing the manuscript.

LITERATURE CITED

Candela, M. E., C. Egea, M. D. García-Pérez, J. Costa, and M. Candela. 2000. Breeding papryka type peppers resistant to Phytophthora capsici. Acta Hortic. 522: 79-86.         [ Links ]

Choi, D., B. L. Ward, and R. M. Bostock. 1992. Differential induction and suppression of potato 3-Hydroxy-3-Methylglutaryl coenzime A reductase genes in response to Phytophthora infestans and to its elicitor arachidonic acid. Plant Cell 4: 1333-1344.         [ Links ]

Erwin, D. C., and O. K. Ribeiro. 1996. Phytophthora Diseases Worldwide. The American Phytopathological Society St. Paul, Minnesota. 562 p.         [ Links ]

Fernández-Herrera, E., R. I. Rojas-Martínez, L. Guevara-Olvera, M. E. Rivas-Dávila, E. Valadez-Moctezuma, y E. Zavaleta-Mejía. 2012. Defensa en chile CM-334 inoculado con Phytophthora capsici e infectado por Nacobbus aberrans. Nematropica 42: 96-107.         [ Links ]

Godínez-Vidal, D., M. Soto-Hernández, M. Rocha-Sosa, E. Lozoya-Gloria, R. I. Rojas-Martínez, L. Guevara-Olvera, y E. Zavaleta-Mejía. 2010. Contenido de capsidiol en raíces de chile CM-334 infectadas por Nacobbus aberrans y su efecto en juveniles del segundo estadio. Nematropica 40: 227-237.         [ Links ]

Ha, S. H., J. B. Kim, Y. S. Hwang, and S. W. Lee. 2003. Molecular characterization of three 3-hydroxy-3-methylglutaryl-CoA reductase genes including pathogen-induced Hmg2 from pepper (Capsicum annuum). Biochim. Biophys. Acta 1625: 253-260.         [ Links ]

Ithal, N., J. Recknor, D. Nettleton, T. Maier, T. J. Baum, and M. G. Mitchum. 2007. Developmental transcript profiling of cyst nematode feeding cells in soybean roots. Mol. Plant-Microbe Interact. 20: 510-525.         [ Links ]

Jammes, F., P. Lecomte, J. A. Engler, F. Bitton, M. L. Martin-Magniette, J. P. Renou, P. Abad, and B. Favery. 2005. Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsis. Plant J. 44: 447-458.         [ Links ]

López-Martínez, N., M. T. Colinas-León, C. B. Peña-Valdivia, Y. Salinas-Moreno, P. Fuentes-Montiel, M. Biesaga, and E. Zavaleta-Mejía. 2011. Alterations in peroxidase activity and phenylpropanoid metabolism induced by Nacobbus aberrans Thorne and Allen, 1944 in chilli (Capsicum annuum L.) CM-334 resistant to Phytophthora capsici Leo. Plant Soil 338: 399-409.         [ Links ]

Manzanilla-López, R. H., M. A. Costilla, M. Doucet, J. Franco, R. N. Inserra, P. S. Lehman, I. Cid del Prado-Vera, R. M. Souza, and K. Evans. 2002. The genus Nacobbus Thorne & Allen, 1944 (Nematoda: Pratylenchidae): Systematics, distribution, biology and management. Nematropica 32: 150-227.         [ Links ]

Ohyama, K., M. Suzuki, K. Masuda, S. Yoshida, and T. Muranaka. 2007. Chemical phenotypes of the hmg1 and hmg2 mutants of Arabidopsis demonstrate the In-planta role of HMG-CoA reductase in triterpene biosynthesis. Chem. Pharm. Bull. 55: 1518-1521.         [ Links ]

SAS Institute Inc. 2002. SAS Procedures Guide, Version 9.0 (Computer program). SAS Institute Inc, Cary, NC.         [ Links ]

Schmittgen, T. D., and K. J. Livak. 2008. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3: 1101-1108.         [ Links ]

Silvar, C., F. Merino, and J. Díaz. 2008. Differential activation of defense-related genes in susceptible and resistant pepper cultivars infected with Phytophthora capsici. J. Plant Physiol. 165: 1120-1124.         [ Links ]

Trujillo-Viramontes, F., E. Zavaleta-Mejía, R. I. Rojas-Martínez, y J. R. Lara. 2005. Tiempo de inoculación y nivel de inóculo, factores determinantes para el rompimiento de resistencia a Phytophthora capsici inducido por Nacobbus aberrans en chile. Nematropica 35: 37-44.         [ Links ]

Ueeda, M., M. Kubota, and K. Nishi. 2006. Contribution of jasmonic acid to resistance against Phytopthora blight in Capsicum annuum cv. SCM334. Physiol. Mol. Plant Pathol. 67: 149-154.         [ Links ]

Weissenborn, D. L., C. J. Denbow, M. Laine, S. S. Lang, Z. Yang, X. Yu, and C. L. Cramer. 1995. HMG-CoA reductase and terpenoid phytoalexins: Molecular specialization within a complex pathway. Physiol. Plant. 93: 393-400.         [ Links ]