Fusarium wilt infection in melon: a transcriptomic approach to characterize the genetic dialogue between host and pathogen 1

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1 Fusarium wilt infection in melon: a transcriptomic approach to characterize the genetic dialogue between host and pathogen 1 K. Szafranska 3, F. Fusari 2, L. Luongo 1, A. Ferrarini 3, A. Polverari 4, M. Delledonne 3, N. Ficcadenti 2, S. Sestili 2, and A. Belisario 1* 1 C.R.A. - Centro di Ricerca per la Patologia Vegetale (CRA-PAV), Via C. G. Bertero, 22, Roma, Italy 2 C.R.A. - Unità di Ricerca per l Orticoltura (CRA-ORA), Via Salaria 1, Monsampolo del Tronto (AP), Italy 3 Dipartimento Scientifico e Tecnologico, Strada Le Grazie 15, Verona, Italy 4 Dipartimento di Scienze, Tecnologie e Mercati della Vite e del Vino, Villa Ottolini Lebrecht, San Floriano di Valpolicella (VR), Italy * Corresponding author alessandra.belisario@entecra.it Keywords: Fusarium oxysporum f. sp. melonis, resistance, Cucumis melo, soilborne pathogen, virulence Abstract Fusarium oxysporum f. sp. melonis (Fom) causes Fusarium wilt of melon which is an economically important disease worldwide. The most effective control measure to prevent Fusarium wilt is through host resistance. Four races of Fom are presently known (0, 1, 2, and 1.2), one of which, race 1.2, is able to overcome the resistance of commonly cultivated varieties. No genes have been identified in melon that confer high levels of resistance to race 1.2. A transcriptomic approach has been undertaken by cdna-aflp on melon plants of the cultivar Charentais Fom-2 infected with race 1 (avirulent) and race 1.2 (virulent), at five time points, and the experiment was concluded 21 days after inoculation. The RNA from fungal colonies of the two races was also included into the analysis, to improve the identification of possible fungal transcripts expressed in the host during infection. Preliminary analysis of expression profiles brought to 500 differentially expressed bands which were selected as corresponding to: a) genes modulated in the incompatible interaction or b) in the compatible interaction only; c) genes modulated in both interactions with different profiles; d) genes expressed in plant, but showing a band of similar size also in the fungal samples, which might be of fungal origin. Fragments of cdna eluted from the gels and sequenced, were searched for homology in databases. Differences in gene expression have been detected between cdna-aflp profiles of virulent and avirulent races grown in culture, which represent the basis for races 1 and 1.2 characterization. INTRODUCTION Fusarium oxysporum f. sp. melonis (Leach & Currence) Snyd. & Hans., causes Fusarium wilt of melon (Cucumis melo L.) which is an economically important disease worldwide. This vascular wilt disease is one of the most difficult adversities to control, because the pathogen is soil-borne and remains viable in the soil on 1 Cucurbitaceae 2008, Proceedings of the IX th EUCARPIA meeting on genetics and breeding of Cucurbitaceae (Pitrat M, ed), INRA, Avignon (France), May th,

2 nonhost crop residues and roots grown in rotation or as chlamydospores for decades (Gordon et al. 1989). The most effective control measure to prevent Fusarium wilt is through host resistance. Four races of F. oxysporum f. sp. melonis are presently known 0, 1, 2, and 1.2, based on the interactions between major host resistance genes and variants of the pathogen (Risser et al. 1976). Race 0 incites disease on those melon genotypes that lack genes for resistance. The two dominant genes Fom-1 and Fom-2 are independently inherited in muskmelon and they control resistance to races 0 and 2, and races 0 and 1, respectively. The presence of both genes in a plant confers a high level of resistance to races 0, 1, and 2. Race 1.2 is able to overcome the resistance of commonly cultivated varieties. Partial resistance to race 1.2 has been found in several Far-Eastern accessions that allowed the breeding of the line Isabelle (Perchepied and Pitrat 2004), and the two double-haploid lines Nad-1 and Nad-2 (Ficcadenti et al. 2002). Although, many Fusarium spp. can penetrate into the cortical tissue of the root, the compatible strains are the only ones able to penetrate the vascular elements. No information is at the moment available on differentially expressed resistance/virulence genes involved in the infectious process of Fusarium wilt race 1 and race 1.2 in melon. The aim of the present work was to investigate on the interaction between the pathogen and the host through the analysis of differentially expressed genes in the vascular colonization by using the approach of cdna-aflp. With this purpose, F. oxysporum f. sp. melonis race 1 and race 1.2 were inoculated on the differential host cultivar Charentais Fom-2. MATERIALS AND METHODS Plant material A total of 150 plants of Charentais Fom-2, at the 4 th -5 th true leaf were utilized for inoculation. Seedlings were grown in trays of 40 plants, in sterilized peat soil mixture, in greenhouse conditions at a temperature C with 16 h of light. Source of inoculum Race 1 and race 1.2w isolates were chosen from the F. oxysporum f. sp. melonis collection of the Centro di Ricerca per la Patologia Vegetale (CRA-PAV) for their high virulence (Belisario et al. 2000). Race designation of the isolates was previously carried out on the basis of the reaction observed on the inoculated differentials, according to the nomenclature proposed by Risser et al. (1976) namely, Charentais Fom-1 resistant to race 0 and race 2, Charentais Fom-2 resistant to race 0 and race 1, and Margot which carries both the resistance genes Fom-1 and Fom-2. Inoculation technique Colonies of F. oxysporum f. sp. melonis were maintained on potato-dextrose agar (PDA, Oxoid) in slant cultures at 4 ± 2 C to obtain new working cultures. Fourteen-day-old cultures of F. oxysporum f. sp. melonis grown at 24 C, were flooded with sterile distilled water and scraped with a sterile glass rod to obtain a spore suspension. This slurry was filtered, and the filtrate was diluted with sterile distilled water to obtain a concentration of 1 x 10 6 conidia (macroconidia and microconidia) ml -1. Seeds of plants to be inoculated were surface-disinfected with 1 % NaOCl for 20 min, rinsed in sterile distilled water, and planted into cell-type 616

3 plastic growing trays filled with a sterilized potting mix of peat and sand (1:1, v/v) (Ficcadenti et al. 2002). Charentais Fom-2 plants were inoculated at the appropriate stage. Seedlings were removed from trays and roots were washed in tap water, about 1 cm pruned, and dipped for 30 min into the spore suspension The inoculated seedlings were transplanted into plastic growing trays (one plant per cell) filled with a steamed potting mix (1 part each of soil:sand:peat) and kept in greenhouse at C, with 16 h of light. The roots of control plants were pruned and dipped in water prior to transplanting. Material submitted to cdna-aflp analysis After inoculation, seedlings were monitored for the extension of the colonization of the pathogen into the inoculated plants by reisolations, and by the recording of symptoms development. The experiment was considered finished after 21 days from the inoculation, when all the plants for the compatible interaction ( Charentais Fom-2 inoculated with race 1.2) displayed obvious and severe symptoms. The incompatible interaction ( Charentais Fom-2 inoculated with race 1) never displayed Fusarium wilt symptoms. On the basis of the results obtained with reisolations (data not shown) and on symptoms occurrence, 5 time points were chosen included time 0 representing the control, and the 21 days after inoculation representing the end of the experiment. The RNA extracted from fungal cultures of both races grown on PDA at 24 C in darkness was also added to the cdna-aflp analysis. For each time point and fungal isolate, the stem of 6 plants was dehydrated in liquid nitrogen and stored in falcon tubes at -80 C for RNA extraction. cdna-aflp analysis The cdna-aflp protocol applied is a modification of the original technique (Breyne et al. 2003) which permits the visualization of one single cdna fragment for each messenger originally present in the sample. The cdna was digested with BstYI and the 3 ends of the fragments were captured on streptavidin magnetic beads (Dynal). Digestion with MseI yielded fragments that were ligated to adapters for amplification. Pre-amplification was performed with an MseI primer combined with a BstYI primer carrying either a T or a C at the 3 end. After preamplification, the mixture was used for selective amplifications with an initial 64 primer combinations carried out with one selective nucleotide added on the 33 P-labeled BstYI primer and two selective nucleotides on the MseI primer. Selective amplification products were separated on a 6 % polyacrylamide gel in a Sequi-Gen GT Sequencing Cell (Bio-Rad). The bands of interest were cut from the gels, eluted in sterile distilled water and used as a template for reamplification using non-labeled primers identical to those employed for selective AFLP amplification. PCR products are purified with MultiScreen PCRµ96 plates (Millipore) and sequenced directly (BMR Genomics). Sequence analysis Bands were selected for the following expression profiles: a) genes modulated in the incompatible interaction or b) in the compatible interaction only; c) genes modulated in both interactions with different profiles; d) genes expressed in plant, but showing a band of similar size also in the fungal samples, which might be of fungal origin. Homology searching by BLAST (Altschul et al. 1997) was carried out against 617

4 the following databases: NCBI ( Melon EST unigene ver 1.0 ( and Fusarium group database: ( group/multihome.html). RESULTS AND DISCUSSION The analysis of cdna-aflp was carried out on RNA samples of infected stems of Charentais Fom-2 which displayed the absence of symptoms until 8 days after inoculation for F. oxysporum f. sp. melonis race 1.2. Plants inoculated with race 1 remained symptomless until the end of the experiment. About 3,500 transcripts were at the moment analyzed, and 500 bands were found differentially expressed. Among those, 14 are melon sequences specifically expressed during the incompatible interaction, and about 300 are specifically expressed only in the late stages of the compatible interaction. As expected, transcripts of the pathogen were abundant in the late phase of infection in the compatible interaction. Approximately 50 fragments of cdna, deriving from infected plants, but showing homology to Fusarium sequences in database, have been identified up to now, some of which are not expressed by the fungus when grown in culture, and might, therefore, represent interesting putative factors involved in pathogenicity. As about the transcripts of F. oxysporum f. sp. melonis colonies grown in vitro, over 20 fragments with race-specific expression were selected, and which represent a source for further investigations for the detection of race 1 and race 1.2. Moreover, over 30 fragments were found differentially expressed between race 1 and race 1.2 that may represent a basis for speculations on the identification of the determinants of virulence. CONCLUSIONS This study represents a new approach for the understanding of the differences in the infectious process of race 1 and race 1.2 of F. oxysporum f. sp. melonis on melon, and for the discovery of genes putatively involved in the response of the host both in the compatible and incompatible interaction. Some promising results, exploitable for race 1 and race 1.2 diagnosis that could allow to overcome the timeconsuming race determination tests, were produced. ACKNOWLEDGEMENTS This research was granted by the project Indagini sui meccanismi di virulenza/resistenza alla Fusariosi per la selezione e costituzione di varietà resistenti di melone (FUSA.MELO.) financed by the Italian Ministry of Agriculture MiPAAF. Literature cited Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: Belisario A, Luongo L, Corazza L, Gordon TR (2000) Indagini su popolazioni di Fusarium oxysporum f. sp. melonis in Italia. Colture Protette 3:

5 Breyne P, Dreesen R, Cannoot B, Rombaut D, Vandepoele K, Rombauts S, Vanderhaeghen R, Inzé D, Zabeau M (2003) Quantitative cdna-aflp analysis for genome-wide expression studies. Mol Genet Genomics 269: Ficcadenti N, Sestili S, Annibali S, Campanelli G, Belisario A, Maccaroni M, Corazza L (2002) Resistance to Fusarium oxysporum f. sp. melonis race 1,2 in muskmelon lines Nad- 1 and Nad-2. Plant Dis 86: Gordon TR, Okamoto D, Jacobson DJ (1989) Colonization of muskmelon and nonsusceptible crops by Fusarium oxysporum f. sp. melonis and other species of Fusarium. Phytopathology 79: Perchepied L, Pitrat M (2004) Polygenic inheritance of partial resistance to Fusarium oxysporum f. sp. melonis race 1.2 in melon. Phytopathology 94: Risser G, Banihashemi Z, Davis, DW (1976) A proposed nomenclature of Fusarium oxysporum f. sp. melonis races and resistance genes in Cucumis melo. Phytopathology 66:

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