Suthanthiram BACKIYARANI, Subbaraya UMA, Marimuthu Somasundram SARASWATHI, Asoor Santhanam SARAVANAKUMAR, Raman THANGAVELU, Viswanathan SRIRAM

Preliminary analysis on the transcripts involved in resistance responses to eumusae leaf spot disease of banana caused by Mycosphaerellaeumusae, a recent add-on of the sigatoka disease complex

Eumusae leaf spot disease, caused by Mycosphaerella eumusae, is widely distributed in different banana growing countries in Asia and Africa, causing severe losses in yield and quality of banana. The aim of this study was to identify the differentially expressed defense responsive genes through subtractive suppression hybridization (SSH) in the Musa M. eumusae interaction system. In this method, leaf samples collected at different intervals between 0 and 72 h postinoculation (hpi) of M. eumusae from resistant (Manoranjitham-AAA) and susceptible (Grand Naine-AAA) cultivars were subjected to cDNA-SSH library construction. From the SSH library, 832 clones having the insert were selected and sequenced. All these sequences were assembled using CAP3, which resulted in 498 unigenes (59.85%), consisting of 78 contigs and 420 singletons. Furthermore, the BLAST2GO analysis performed showed that, out of the 498 unigenes, only 161 (32.32%) were involved in molecular functions, cellular component and biological processes. However, among these 161 unigenes, 57 were found to have top BLAST hits. Six genes were selected based on the function and subjected to validation through qPCR. Four genes, namely ethylene responsive factor, flavin containing monoxygenase, serine glyoxylate, and metallothionein, were found to have the highest-fold regulation in the resistant cultivar at 48 hpi, followed by lipoxygenase at 36 hpi. The main findings are that resistant plants deploy HR activity as a strategy to evade the fungus followed by the activation of various ROS scavengers to maintain homeostasis and the role of phytohormones may be induced as a part of plant defense against M. eumusae.

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Arzanlou M, Abeln EC, Kema GH, Waalwijk C, Carlier J, Vries I, Guzmán M, Crous PW (2007). Molecular diagnostics for the sigatoka disease complex of banana. Phytopathology 97: 1112-1118.
Aguilar-Barragan A, García-Torres AE, Odriozola-Casas O, Macedo-Raygoza G, Ogura T, Manzo-Sánchez G, James AC, Islas-Flores I, Beltrán-García MJ (2014). Chemical management in fungicide sensitivity of Mycosphaerella fijiensis collected from banana fields in México. Braz J Microbiol 45: 359-364.
Backiyarani S, Uma S, Arunkumar G, Saraswathi MS, Sundararaju P (2014). Differentially expressed genes in incompatible interactions of Pratylenchus coffeae with Musa using suppression subtractive hybridization. Physiol Mol Plant P 86: 11-18.
Barcelo AR, Laura VGR (2009). Reactive oxygen species in plant cell walls. In: Rio LA, Puppo A, editors. Reactive Oxygen Species in Plant Signaling. Berlin, Germany: Springer, pp. 73-93.
Beltran-Garcia MJ, Manzo-Sanchez G, Guzman-Gonzalez S, Arias-Castro C, Rodriguez-Mendiola M, Avila-Miranda M, Ogura T (2009). Oxidative stress response of Mycosphaerella fijiensis, the causal agent of black leaf streak disease in banana plants, to hydrogen peroxide and paraquat. Can J Microbiol 55: 887-894.
Berrocal-Lobo M, Molina A, Solano R (2002). Constitutive expression of ethylene-response-factor 1 in Arabidopsis confers resistance to several necrotrophic fungi. Plant J 29: 23-32.
Binks RH, Greenham JR, Luis JG, Gowen SR (1997). A phytoalexin from roots of Musa acuminata var. Pisang sipulu. Phytochemistry 45: 47-49.
Carlier J, Zapater MF, Lapeyre F, Jones DR, Mourichon X (2000). Septoria leaf spot of banana: a newly discovered disease caused by Mycosphaerella eumusae (anamorph Septoria eumusae). Phytopathology 90: 884-890.
Chen L, Zhong HY, Kuang JF, Li JG, Lu WJ, Chen JY (2011).Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions. Planta 234: 377-390.
Conesa A, Götz S, Garcia-Gomez JM, Terol J, Talon M, Robles M (2005). Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21: 3674-3676.
Crous PW, Mourichon X (2002). Mycosphaerella eumusae and its anamorph Pseudocercospora eumusae spp. nov.: causal agent of eumusae leaf spot disease of banana. Sydowia 54: 35-43.
DHont A, Denoeud F, Aury JM, Baurens FC, Carreel F, Garsmeur O, Noel B, Bocs S, Droc G, Rouard M et al. (2012). The banana (Musa acuminata) genome and the evolution of monocotyledonous plants. Nature 488: 213-217.
Das K, Roychoudhury A (2014). Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants. Front Environ Sci 2: 1-13.
Exotic Pest Alert: Eumusae leaf spot (2014). Primefact 1329, 1st edition Plant Biosecurity & Product Integrity, Orange.
Food and Agriculture Organization of the United Nations, FAOSTAT database (2013), available at http://faostat3.fao.org/browse/Q/*/E.
Gasic K, Hernandez A, Korban SS (2004). RNA extraction from different apple tissues rich in polyphenols and polysaccharides for cDNA library construction. Plant Mol Biol Rep 22: 437a-437g.
Hadrami AE, Kone D, Lepoivre P (2005). Effect of juglone on active oxygen species and antioxidant enzymes in susceptible and partially resistant banana cultivars to Black Leaf Streak Disease. Eur J Plant Pathol 113: 241-254.
Hidalgo W, Jima NC, Riya CM, Felipe O, Bernd S (2015). Phenylphenalenones protect banana plants from infection by Mycosphaerella fijiensis and are deactivated by metabolic conversion. Plant Cell Environ. doi: 10.1111/pce.12630.
Hölscher D, Dhakshinamoorthy S, Alexandrov T, Becker M, Bretschneider T, Buerkert A, Crecelius AC, De Waele D, Elsen A, Heckel DG et al. (2014). Phenalenone-type phytoalexins mediate resistance of banana plants (Musa spp.) to the burrowing nematode Radopholus similis. P Natl Acad Sci USA 111: 105-110.
Jones RD (2002). The distribution and importance of the Mycosphaerella leaf spot diseases of banana. Proceeding. 2nd International. Workshop on Mycosphaerella Leaf Spot Disease of Bananas. INIBAP, Montpellier, France: 25-42.
Lorenzo O, Piqueras R, Sanchez-Serrano JJ, Solano R (2003). ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense. Plant Cell 15: 165-178.
Luis JG, Lahlou EH, Andrés LS, Echeverri F, Fletcher WQ (1997). Phenylphenalenonic phytoanticipins. New acenaphtylene and dimeric phenylphenalenones from the resistant Musa selected hybrid SH-3481. Tetrahedron 53.
Luis JG, Quinones W, Echeverri F, Grillo TA, Kishi MP, Garcia-Garcia F, Torres F, Cardona G (1996). Musanolones: Four 9-phenylphenalenones from rhizomes of Musa acuminata. Photochemistry 41: 753-757.
Maria de Jesus BC, Jacques E, Jean PM, Rocio ER, Michel RN, Luis CO, Chantal H, Marc L, Jean LV (2011). Reactive oxygen species and cellular interactions between Mycosphaerella fijiensis and banana. Trop Plant Biol 4: 134-143.
National Horticulture Board NHB (2013), available at http://www.nhb.gov.in.
Passos MA, de Cruz VO, Emediato FL, de Teixeira CC, Azevedo VC, Brasileiro AC, Amorim EP, Ferreira CF, Martins NF, Togawa RC et al. (2013). Analysis of the leaf transcriptome of Musa acuminata during interaction with Mycosphaerellamusicola: gene assembly, annotation and marker development. BMC Genomics 14: 78.
Portal O, Izquierdo Y, De VlD, Sánchez RA, Mendoza RM, Acosta SM, Ocaña B, Jiménez E, Höfte M (2011). Analysis of expressed sequence tags derived from a compatible Mycosphaerella fijiensis-banana interaction. Plant Cell Rep 30: 913-28.
Rivas GG, Zapater MF, Abadie C, Carlier J (2004). Founder effects and stochastic dispersal at the continental scale of the fungal pathogen of bananas Mycosphaerella fijiensis. Mol Ecol 13: 471-482.
Slaymaker DH, Navarre DA, Clark D, Del Pozo O, Martin GB, Klessig DF (2002).The tobacco salicylic acid-binding protein 3 (SABP3) is the chloroplast carbonic anhydrase, which exhibits antioxidant activity and plays a role in the hypersensitive defense response. P Natl Acad Sci 99: 11640-11645.
Solano R, Stepanova A, Chao Q, Ecker JR (1998). Nuclear events in ethylene signaling: A transcriptional cascade mediated by ethylene-insensitive3 and ethylene response- factor1. Gene Dev 12: 3703-3714.
Stierle AA, Upadhyay R, Hershenhorn J, Strobel GA, Molina G (1991). The phytotoxins of Mycosphaerella fijiensis, the causative agent of Black Sigatoka disease of bananas and plantains. Experientia 47: 853-859.
Swarupa V, Ravishankar KV, Rekha A (2013).Characterization of tolerance to Fusarium oxysporum f.sp., cubense infection in banana using suppression subtractive hybridization and gene expression analysis. Physiol Mol Plant P 83: 1-7.
Thangavelu R, Ganga Devi P, Gopi M, Mustaffa MM (2013). Management of Eumusae leaf spot disease of banana caused by Mycosphaerella eumusae with Zimmu Allium Sativum x Allium cepa) leaf extract. Crop Prot 46: 100-105.
Van Den Berg N, Berger DK, Hein I, Birch PR, Wingfield MJ, Viljoen A (2007). Tolerance in banana to Fusarium wilt is associated with early up-regulation of cell wall-strengthening genes in the roots. Mol Plant Pathol 8:333-41.
Van der Hoorn RA, Jones JD (2004).The plant proteolytic machinery and its role in defense. Curr Opin Plant Biol 7: 400-407.
Xia QZ, Zhang XL, Nie YC, Guo XP (2005). Withdrawal of exogenous auxin induces programmed cell death of cotton embryonic suspension cultures. Journal of Plant Physiology and Molecular Biology 31: 78-84.
Zinoveva SV, Udalova ZV, Vasileva IS, Vanyushkin SA, Paseshnichenko VA (2001). Role of isoprenoids in plant adaptation to biogenic stress induced by parasitic nematodes. Appl Biochem Micro 37: 456-463.