Abstract
A novel zinc‐binding alcohol dehydrogenase 2 (AdZADH2) was significantly upregulated in a wild peanut, Arachis diogoi treated with conidia of late leaf spot (LLS) pathogen, Phaeoisariopsis personata. This upregulation was not observed in a comparative analysis of cultivated peanut, which is highly susceptible to LLS. This zinc‐binding alcohol dehydrogenase possessed a Rossmann fold containing NADB domain in addition to the MDR domain present in all previously characterized plant ADH genes/proteins. Transient over‐expression of AdZADH2 under an estradiol inducible promoter (XVE) resulted in hypersensitive response (HR)‐like cell death in tobacco leaf. However, the same level of cell death was not observed when the domains were transiently expressed individually. Cell death observed in tobacco was associated with overexpression of cell death related proteins, antioxidative enzymes such as SOD, CAT and APX and pathogenesis‐related (PR) proteins. In A. diogoi, AdZADH2 expression was significantly upregulated in response to the plant signaling hormones salicylic acid, methyl jasmonate, and sodium nitroprusside.
Highlights
A novel zinc-binding alcohol dehydrogenase 2 (AdZADH2) was significantly upregulated in a wild peanut, Arachis diogoi treated with conidia of late leaf spot (LLS) pathogen, Phaeoisariopsis personata
A partial fragment of a zinc-binding alcohol dehydrogenase 2 was differentially upregulated in a cDNA-AFLP analysis of an interaction between Arachis diogoi and the late leaf spot pathogen P. personata [17]
The NADB Rossmann domain was absent in other plant alcohol dehydrogenases (ADH) proteins studied so far (Fig. S4) showing that it is a novel ADH2
Summary
50/30 RACE-PCR, isolation of full length cDNA and transient conditional overexpression of AdZADH2. Rapid amplification of cDNA ends (RACE) was performed to clone the full length cDNA of AdZADH2 by using SMARTTM RACE cDNA Amplification kit (Clontech, California, USA) using gene specific primers designed from the sequence of a partial fragment identified in an earlier study using cDNA-AFLP [17]. The ORF was amplified with primers AdZADH2-ApaI-F and AdZADH2-SpeI-R containing ApaI and SpeI restriction enzymes, respectively, by using PhusionTM DNA polymerase (Finnzymes, Loughborough, UK, NEB, Table S1), cloned in pTZ57R/T and sequenced. Total RNA was isolated from leaf samples with and without estradiol treatment collected at 0, 24, and 48 h time points. CDNA samples were diluted fivefold and 0.5 lL of the diluted reaction mixture was taken as qRT-PCR template in a 20 lL total reaction volume containing 0.4 lM gene specific primers with lL SYBR Premix Ex Taq with ROX (Takara Bio Inc., Kusatsu, Shiga, Japan) and the samples were appraised in three technical replicates. Relative fold change in RNA expression was estimated using DDCT method [24]
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