Transcriptomic and Proteomic Analyses of Resistant Host Responses in Arachis diogoi Challenged with Late Leaf Spot Pathogen, Phaeoisariopsis personata

Abstract

Late leaf spot is a serious disease of peanut caused by the imperfect fungus, Phaeoisariopsis personata. Wild diploid species, Arachis diogoi. is reported to be highly resistant to this disease and asymptomatic. The objective of this study is to investigate the molecular responses of the wild peanut challenged with the late leaf spot pathogen using cDNA-AFLP and 2D proteomic study. A total of 233 reliable, differentially expressed genes were identified in Arachis diogoi. About one third of the TDFs exhibit no significant similarity with the known sequences in the data bases. Expressed sequence tag data showed that the characterized genes are involved in conferring resistance in the wild peanut to the pathogen challenge. Several genes for proteins involved in cell wall strengthening, hypersensitive cell death and resistance related proteins have been identified. Genes identified for other proteins appear to function in metabolism, signal transduction and defence. Nineteen TDFs based on the homology analysis of genes associated with defence, signal transduction and metabolism were further validated by quantitative real time PCR (qRT-PCR) analyses in resistant wild species in comparison with a susceptible peanut genotype in time course experiments. The proteins corresponding to six TDFs were differentially expressed at protein level also. Differentially expressed TDFs and proteins in wild peanut indicate its defence mechanism upon pathogen challenge and provide initial breakthrough of genes possibly involved in recognition events and early signalling responses to combat the pathogen through subsequent development of resistivity. This is the first attempt to elucidate the molecular basis of the response of the resistant genotype to the late leaf spot pathogen, and its defence mechanism.

Variability and Correlations among Groundnut Populations for Early Leaf Spot, Pod Yield, and Agronomic Traits

“…Among the main foliar fungal diseases, early leaf spot (ELS) caused by Cercospora arachidicola Hori and late leaf spot (LLS) caused by Phaeoisariopsis personata (Bert and Curtis) Deigton are the more destructive diseases [4,5]. The two diseases combined can cause pod and fodder yield loss of over 50% to 70% [6,7].…”

A novel zinc‐binding alcohol dehydrogenase 2 from Arachis diogoi, expressed in resistance responses against late leaf spot pathogen, induces cell death when transexpressed in tobacco

“…AdZADH2 sequence analysis 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]. It was made full length using 3 0 /5 0 RACE.…”

“…Rapid amplification of cDNA ends (RACE) was performed to clone the full length cDNA of AdZADH2 by using SMART ™ 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 Apa I and Spe I restriction enzymes, respectively, by using Phusion ™ DNA polymerase (Finnzymes, Loughborough, UK, NEB, Table S1), cloned in pTZ57R/T and sequenced.…”

“…The extent of cell death was quantified by Evans Blue staining following Baker and Mock [21] method. For electrolyte leakage analysis, leaf discs (8 mm diameter) were cut off from agro-infiltrated regions after 48 h post infiltration using a cork borer and transferred to distilled water containing 30 lM 17b-estradiol in Petri dishes under bright light with 14/ 10 h photoperiod [17,22,23]. Electrolyte leakage values were determined by using an electro-conductivity meter (Digisun Electronics, Hyderabad, India) at different time intervals and data were plotted from three biological replicates.…”

“…Several wild relatives of peanut including Arachis diogoi are highly resistant to many foliar diseases and biotic stresses [16]. In an earlier investigation, we have identified a Zinc-binding alcohol dehydrogenase 2 (AdZADH2) gene in Arachis diogoi in its interaction with Phaeoisariopsis personata that causes the serious late leaf spot disease in the cultivated peanut, Arachis hypogaea [17]. In this communication, we have focused on the cloning of the novel AdZADH2 and its involvement in HR-mediated cell death in tobacco.…”