Defence Transcriptome Profiling of Zingiber Zerumbet (L.)
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Defence transcriptome profi ling in Z. zerumbet 81 Defence transcriptome profi ling of Zingiber zerumbet (L.) Smith by mRNA differential display P G KAVITHA and GEORGE THOMAS* Plant Molecular Biology Group, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695 014, India *Corresponding author (Fax, 91-471-2348096; Email, [email protected]) Soft rot is a serious disease in ginger (Zingiber offi cinale Roscoe), imposing a considerable economic loss annually in all ginger-producing countries. In this study, mRNA differential display was employed to identify genes whose expression was altered in a soft rot-resistant accession of Zingiber zerumbet (L.) Smith, a wild relative of ginger, in response to Pythium aphanidermatum (Edson) Fitzp., which is the principal causative agent of soft-rot disease in ginger. Analysis using 68 primer combinations identifi ed 70 differentially expressed transcript-derived fragments (TDFs), of which 34 TDFs were selected for further analysis following reverse northern screening. Cloning and sequence characterization of the 34 TDFs yielded a total of 54 distinct clones. Functional categorization of these clones revealed seven categories, of which the defence/stress/signalling group was the largest, with clones homologous to genes known to be actively involved in various pathogenesis-related functions in other plant species. The signifi cance of these genes in relation to the resistance response in Z. zerumbet is discussed. This study has provided a pool of candidate genes for detailed molecular dissection of the defence mechanisms in Z. zerumbet and for accessing wild genetic resources for the transgenic improvement of ginger. [Kavitha P G and Thomas G 2008 Defence transcriptome profi ling of Zingiber zerumbet (L.) Smith by mRNA differential display; J. Biosci. 33 81–90] 1. Introduction the rhizomes, causing the inner tissues to decay, and ends in wilting and complete drying of the plant (Selvan et al Ginger (Zingiber offi cinale Roscoe) is a herbaceous 2002). perennial belonging to the tropical monocotyledonous To date, no effective fungicides or biological control family Zingiberaceae. The thick, scaly, aromatic rhizome methods are available for controlling Pythium species (underground stem) of the ginger plant is valued the world (Folman et al 2003). Ginger is an obligate asexual over both as a spice and as a medicine. India is the second- reproducer, propagated exclusively through rhizomes. largest producer of ginger in the world, and contributed to Conventional crop improvement methods involving sexual one-fi fth (230 000 metric tons) of the world production in hybridization are therefore ineffective in ginger. Moreover, 2005 (http://faostat.fao.org/site/291/default.aspx). Soft-rot genetic variation for resistance to soft rot is not present in the disease caused by different species of the oomycete Pythium ginger germplasm (Dake 1995). Given these impediments, severely affects ginger cultivation in all ginger-producing transgenic improvement using a suitable genetic variation regions of the world (Lawrence 1984). Once this soil-borne from alien resources could be a possible alternative for pathogen makes its appearance in a fi eld, it spreads rapidly the genetic improvement of ginger. With that as a goal, we and kills the entire crop; moreover, the pathogen can survive previously screened wild relatives of ginger for resistance in the soil for years as oospores. Infection begins at the collar to P. aphanidermatum (Edson) Fitzp., the most prevalent region of the pseudostem of the ginger plant and spreads into causative agent of soft-rot disease (Selvan et al 2002), and Keywords. Differential display; ginger; Pythium aphanidermatum; soft rot; transcript profi ling; Zingiber zerumbet Abbreviations used: CaM, calmodulin; FGAM, formylglycinamidine; MAP, mitogen-activated protein; PCR, polymerase chain reaction; TDF, transcript-derived fragment http://www.ias.ac.in/jbiosci J. Biosci. 33(1), March 2008, 81–90, © IndianJ. Academy Biosci. 33 of(1), Sciences March 2008 81 82 P G Kavitha and George Thomas identifi ed Zingiber zerumbet (L.) Smith as a potential source 2.2 RNA isolation and differential display of resistance (Kavitha and Thomas 2007). It is important to understand the network of genes that For RNA isolation, tissue was collected from pseudostems governs resistance to Pythium in Z. zerumbet in order to inoculated with P. aphanidermatum zoospores at 24 h choose candidate genes for engineering soft-rot resistance post-inoculation (hpi), 48 hpi and 96 hpi. Tissue collected into ginger. On the whole, the Zingiberaceae family is poorly just before inoculation (0 hpi) was used as a control. studied and little is known about the molecular mechanisms RNA was isolated according to Salzmann et al (1999). of host resistance among its species. Though genetic Total RNA was treated with DNase I to remove any determinants governing biotic and abiotic stress resistance traces of genomic DNA present. mRNA differential have been reported in diverse plants (Kim et al 2000; display analysis was carried out according to Liang and Mahalingam et al 2003; Bray 2004; Ameline-Torregrosa Pardee (1992) with modifi cations. Each RNA sample et al 2006), extrapolating such results to taxonomically was subjected to three separate reverse-transcription unrelated Zingiber species may not be appropriate, as the reactions, each with one of the three one-base-anchored molecular events and biochemical processes vary between oligo-dT primers: HT11G (5′ AAGCTTTTTTTTTTTG pathosystems (Ameline-Torregrosa et al 2006). Moreover, 3′), HT11C (5′ AAGCTTTTTTTTTTTC 3′) and HT11A (5′ little is known about the molecular basis of host defence AAGCTTTTTTTTTTTA 3′). Differential display analysis against oomycetes (Kamoun et al 1999; Latijnhouwers was performed on the reverse-transcribed products using et al 2003), especially against the Pythium species, which the corresponding oligo-dT primers and random decamer affect mostly root tissues. We initiated a programme to primers (Operon Technologies, Alameda, USA). The understand the molecular mechanisms governing the random primers used in combination with each anchor resistance of Z. zerumbet to P. aphanidermatum and to primer are given in table 1. The 20 µl reaction medium further study the factors identifi ed thereupon for the contained 50 ng of reverse-transcribed product, 1X PCR improvement of ginger. Here we report defence transcrip- buffer, 25 µM dNTPs, 2 µM random primer, 2 µM anchor tome profi ling of a resistant accession of Z. zerumbet primer, 0.2 µl of 2000 Ci/mmol α [33P] dATP and 1 u using the mRNA differential display technique to detect Taq DNA polymerase (Qiagen). The polymerase chain genes whose expression is altered in response to P. reaction (PCR) conditions were as follows: 94ºC for 30 s; aphanidermatum. 42ºC for 1 min; 72ºC for 1 min for 25 cycles followed by 72ºC for 5 min in a thermocycler (Mastercycler gradient, Eppendorf). 2. Materials and methods 2.1 Plant materials and pathogen inoculation 2.3 Gel electrophoresis, band elution and reamplifi cation A fi eld isolate of P. aphanidermatum (RGCB P117) The differential display products were fractionated on a 6% obtained from the Indian Institute of Spices Research, denaturing polyacrylamide gel at 45 W for 2.5 h and the Calicut, Kerala, India was used for the inoculation studies. gel was autoradiographed. Differential bands were excised For zoospore production, mycelial discs were cut from the after aligning the autoradiogram with the gel. The excised margins of a two-day-old Pythium culture and transferred gel pieces were rehydrated in 100 µl of sterile water for to a Petri plate containing sterile double-distilled water and 10 min, and the swollen gel pieces were boiled for 15 min boiled pieces of grass leaves as spreading supports. The and centrifuged at 10 000 g for 2 min. The DNA was plates were incubated under direct light for 48 h. The plates precipitated from the supernatant with double the volume of with sporulating mycelia were given a cold shock at 4 °C for absolute alcohol in the presence of 60 mM sodium acetate 10 min to release zoospores from the sporangia. A resistant and 100 µg/ml glycogen for 30 min at –70ºC. The precipitate accession of Z. zerumbet (Acc. no. 2010-9) (Kavitha and was rinsed with 85% ethanol and dissolved in 10 µl sterile Thomas 2007) was used as the host. Rhizomes harvested water. The eluted fragments were reamplifi ed following the from mature plants were sprouted in earthen pots in a red- same PCR conditions used for amplifi cation of the reverse- earth:sand:leaf compost mixture and three-month-old plants transcribed product, but with non-radioactive dNTPs only. were used for inoculation. The inoculation was done by The eluted cDNA fragments were cloned using the pGEM-T pouring 500 µl of the zoospore suspension (2.0 x 106 spores/ Easy Vector System (Promega) and E. coli JM 109 as a host. ml) onto the collar region (region just above the ground) of Plasmid isolation from the transformed colonies was carried the pseudostem. Three pinpricks were given at the collar out using the Wizard Plus SV Miniprep DNA Purifi cation region before inoculation. The plants were maintained in System (Promega). a net house with a misting facility at 27–30ºC and 80–90% The PCR-amplifi ed cDNA fragments eluted from the humidity. differential display gel were referred to as transcript-derived J. Biosci. 33(1), March 2008 Defence transcriptome profi ling in Z. zerumbet 83 fragments (TDFs) and the clones derived from the TDFs as pseudostems (24 hpi, 48 hpi and 96 hpi), were examined cDNA clones. visually for the distribution of each band across the samples. Representative differential display profi les are shown 2.4 Reverse northern screening in fi gure 1. Most cDNA bands were of similar intensity in both the control and Pythium-treated materials. These are Reverse northern analysis was carried out on the likely to represent transcripts from housekeeping genes. differentially expressed TDFs and on clones derived from However, some bands were unique to, or expressed in selected TDFs.