Identification of genes expressed during the interaction between cv. Florina and V. inaequalis by differential display-Rt-Pcr

Mariana Mota¹, Cristina Moniz Oliveira¹

RESUMO

Para identificar genes especificamente regulados durante a expressão do mecanismo de resistência ao pedrado na macieira cv. Florina, comparou-se o padrão de expressão genética em plantas após inoculação com uma suspensão conidial do fungo Venturia inaequalis, com o padrão em plantas inoculadas com água, por “differential display RT-PCR”. Os produtos das reacções de amplificação resultantes da combinação de um oligodT (dT_VG) com 20 iniciadores decameros de sequência arbitrária foram separados em gel desnaturante de poliacrilamida e detectados com nitrato de prata. 14 fragmentos de cDNA visíveis apenas na amostra derivada de folhas inoculadas com a suspensão, ou em maior abundância nesta amostra, foram extraídos do gel para purificação, reamplificação e clonagem. Três fragmentos foram já sequenciados, revelando uma sequência forte homologia com uma fitoquelatina sintetase vegetal, potencialmente envolvida em respostas a factores de “stress”. A acumulação diferencial deste cDNA em resposta à inoculação será verificada por transcrição-reversa-PCR com iniciadores específicos.

Palavras-chave: differential-display RTPCR, expressão diferencial, Florina, pedrado, fitoquelatina sintetase

ABSTRACT

In order to identify genes that are specifically regulated during the expression of the resistance mechanism of the apple cv. Florina towards the apple scab fungus, the genetic expression pattern in plants inoculated with a conidial suspension of Venturia inaequalis was compared with the expression pattern in plants inoculated with water, by differential display RT-PCR. Amplified products derived from the combination of one oligodT (dT_VG) with 20 10-mer primers were separated in polyacrylamide denaturing gel and detected with silver nitrate. 14 cDNA fragments that were only visible in the sample inoculated with fungal suspension, or more abundant in this one, were purified from the gel for reamplification and cloning. Three fragments were already sequenced and one sequence showed very strong homology with a plant phytochelatin synthetase, that might be involved in stress responses. Differential accumulation of this cDNA upon fungal inoculation shall be verified by reverse-transcription PCR with specific primers.

Key-words: apple scab, differential-display RT-PCR, differential expression, Florina, phytochelatin synthetase

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Bassam, B.J.; Caetano-Anolles, G. & Gresshoff, P.M. (1991) -Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Biochem. 196: 80-83.         [ Links ]

Basse, C.W. (2005) -Dissecting defense-related and developmental transcriptional responses of maize during Ustilago maydis infection and subsequent tumor formation. Plant Physiology 138: 1774-1784.

Bénaouf, G. & Parisi, L. (1997) -Pathogenicity of Venturia inaequalis strains from Malus floribunda 821: comparison with race 6 on apple clones. IOBC/WPRS Bulletin 20: 8-11.

Benitez, Y.; Botella, M.A.; Trapero, A.; Alsalimiya, M.; Caballero, J.L.; Dorado, G. & Munoz-Blanco, J. (2005) -Molecular analysis of the interaction between Olea europaea and the biotrophic fungus Spilocaea oleagina. Molecular Plant Pathology 6: 425-438.

Benito, E.P.; Prins, T. & van Kan, J.A.L. (1996) - Application of differential display RT-PCR to the analysis of gene expression in a plant-fungus interaction. Plant Mol. Biol. 32: 947-957.

Chang, S.; Puryear, J. & Cairney, J. (1993) - A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. Rep. 11: 113-116.

Chevalier, M.; Lespinasse, Y. & Renaudin, S. (1991) - A microscopic study of the different classes of symptoms coded by the Vf gene in apple for resistance to scab (Venturia inaequalis). Plant Pathol. 40: 249-256.

Chevalier, M. & Lespinasse, Y. (1994) - Ultrastructural study of host-parasite interaction: Early phases of Venturia inaequalis installation on susceptible and resistant apple leaves. Bulletin de l’Association des Anatomistes 78: 33-37.

Collinge, M. & Boller, T. (2001) - Differential induction of two potato genes, Stprx2 and StNAC, in response to infection by Phytophthora infestans and to wounding. Plant. Mol. Biol. 46: 521-529.

Degenhardt, J.; Al-Mari, A.N.; Kurkcuoglu, S.; Szankowski, I. & Gau, A.E. (2005) -Characterization by suppression subtractive hybridization of transcripts that are differentially expressed in leaves of apple scab-resistant and susceptible cultivars of Malus domestica. Mol. Genet. Genomics 273:326-335.

Eckey C. (2002) - Isolierung und Charakterisierung Pathogen-induzierter Gene der Gerste (Hordeum vulgare L.) und Markerentwicklung für den Mlg Resistenzgenlocus mittels cDNA-AFLP. Dissertação de doutoramento, Universidade Justus Lie-big, Giessen, 151 pp.

Fischer, M.; Fischer, C. & Dierend, W. (2005) - Evaluation of the stability of scab resistance in apple: a co-operation between gene bank curator, breeder and fruit grower. PGR Newsletter 142: 36-42.

Fourney, M.R.; Miyakoshi, J.; Day III, R.S.; Paterson, M.C. (1988) - Northern blotting: efficient RNA staining and transfer. Focus 10: 5-7.

Gao, Z.S. & Weg, W.E. (2006) - The Vf gene for scab resistance in apple is linked to sub-lethal genes. Euphytica 151:123-132.

Garcia-Lorenzo, M.; Sjodin A.; Jansson, S. & Funk, C. (2006) - Protease gene families in Populus and Arabidopsis. BMC Plant Biology 6 doi:10.1186/1471-2229-6-30

Ivanicka, J.; Kellerhals, M. & Theiler, R. (1996) - Evaluation of scab (Venturia inaequalis (Cooke) G. Wint.) on shoots and detached leaves from in vitro and greenhouse grown plants of the apple (Malus domestica Mill.) cultivars Golden Delicious and Florina. Gartenbauwissenschaft 61: 242-248.

Kanaoka, M.; Urban, S.; Freeman M. & Okada, K. (2005) -An Arabidopsis Rhomboid homolog is an intramembrane protease in plants. FEBS Letters 579: 5723-5728.

Komjanc, M.; Festi, S.; Rizzotti, L.; Cattivelli, L.; Cervone, F. & De Lorenzo, G. (1999) - A leucine-rich repeat receptor-like protein kinase (LRPKm1) gene is induced in Malus x domestica by Venturia inaequalis infection and salicylic acid treatment. Plant Mol. Biol. 40: 945-957.

Lapopin, L.; Gianinazzi-Pearson, V. & Franken, P. (1999) -Comparative differential RNA display analysis of arbuscular mycorrhiza in Pisum sativum wild type and a mutant defective in late stage development. Plant Mol. Biol. 41: 669-677.

Liang, P. & Pardee, A.B. (1992) - Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: 967-971.

Lievens, S.; Goormachtig, S. & Holsters, M. (2001) - A critical evaluation of differential display as a tool to identify genes involved in legume nodulation: looking back and looking forward. Nucleic Acids Research 29: 3459-3468.

Lawrence, C.B.; Bass, W.T.; Kovar, J. & Qiu, J. (2006) -Profiling of Gene Expression During Early Stages of A Compatible Plant-Oomycete Interaction via cDNA-AFLP® Technology. Disponível em <http://www.licor.com/bio/Posters/536/pdf/536Poster.pdf> (acesso a 19/11/07).

Palmiter, D.H. (1934) - Variability in mono-conidial cultures of Venturia inaequalis. Phytopathology 24: 22-47.

Sambrook, J.; Fritsch, E.F. & Maniatis T. (1989) - Molecular cloning: a laboratory manual. Cold Spring Harbor. Laboratory Press, Cold Spring Harbor.

Yi, T.Y.; Zhang, B.X.; Xie, B.Y. & Gao, B.D. (2005) -Application of differential display techniques to the cloning of resistant gene to phytophthora blight. Journal of Hunan Agricultural University 31: 258-261.

Comunicação apresentada no 5º Congresso da Sociedade Portuguesa de Fitopatologia, Coimbra 2007

Recepção/Reception: 2008.02.19 aceitação/acception: 2008.10.07