Microarray-based screening of differentially expressed genes in peanut in response to Aspergillus parasiticus infection and drought stress

Abstract

Aflatoxin contamination caused by Aspergillus fungi is a great concern in peanut production worldwide. Pre-harvest A. parasiticus infection and aflatoxin contamination are usually severe in peanuts that are grown under drought stressed conditions; however, drought tolerant peanut lines have less aflatoxin contamination. The objective of this study was to identify resistance genes in response to Aspergillus parasiticus infection under drought stress using microarray and real-time PCR. To identify transcripts involved in the resistance, we studied the gene expression profiles in peanut genotype A13 which is drought tolerant and resistant to pre-harvest aflatoxin contamination, using cDNA microarray containing 384 unigenes selected from two expressed sequenced tag (EST) cDNA libraries challenged by biotic and abiotic stresses. A total of 42 up-regulated genes (log₂ ratio > 1) in several functional categories were detected under both A. parasiticus challenge and drought stress. A total of 52 up-regulated genes were detected in response to drought stress alone. There were 25 genes commonly expressed in both treatments. The top 20 up-regulated gene from A. parasiticus challenge and drought stress were selected for validation of their expression levels using real-time PCR. A13 was also used to study the functional analysis of these genes and a possible link of these genes to the resistance trait. Although the selected genes identified by microarray analysis were validated by real-time PCR, further investigations are needed to characterize each of these genes. Gene probes could then be developed for application in breeding selection.

Introduction

Peanut originated in South America and is grown worldwide for human food. Aspergillus flavus and Aspergillus parasiticus can colonize seeds of several agricultural crops including peanut. This can result in contamination of seeds with toxic fungal metabolite aflatoxins. Aflatoxin contamination is a great concern in peanut production because this toxin can cause teratogenic and carcinogenic diseases in animal and human [36], [43]. These fungi are ubiquitous, being found virtually everywhere in the world. They are soil-borne, but prefer to grow on high nutrient media (e.g. seeds). A. flavus appears to be the primary aflatoxin-producing fungus on these commodities, although A. parasiticus also occurs frequently on peanut. Both fungi produce a family of related aflatoxins; the one most commonly produced by A. flavus are B1 and B2, while A. parasiticus produces two additional aflatoxins, G1 and G2. Damage due to environmental stress (drought) can enable the fungi to invade seeds where they thrive at high temperatures and extremely dry conditions, such as those frequently experienced in the Southern U.S. during the summer [46].

Plant stresses, depending on the type of stress and the type of plant, may include factors affecting plant survival, growth and development of seed for harvest [46]. Tremendous efforts have been made by scientists worldwide to study the mechanisms of the environmental factors that affect crop yield [10], [26], [32], [34], [38]. Among these factors, resistance to insects, fungal infection/aflatoxin formation and drought stress are genetic properties of the crop variety.

Although peanut aflatoxin contamination was reported 40 years ago [4], systematic research on the host resistance mechanism against aflatoxin contamination was not possible until the first two resistant peanut genotypes (PI 337394F and PI 337409) were identified [31]. In the 1980s, more resistant genotypes were identified [30], [44], [48] and more research activities were carried out to correlate the relationship of pre-harvest aflatoxin contamination and drought stress. Researchers observed that drought stress could increase fungal infection and aflatoxin contamination [3], [5], [37]. Under drought stress, the loss of the capacity of peanut seeds to produce phytoalexins resulted in higher aflatoxin contamination [14], [47]. The active water of the seeds is the most important factor controlling the capacity of seeds to produce phytoalexins. However, mature peanuts possessed additional resistance to aflatoxin contamination that could not be attributed solely to phytoalexin production [14]. Based on the understanding that drought stress increases pre-harvest aflatoxin contamination, drought tolerant lines were speculated to possess some degree of resistance to aflatoxin contamination [19]. Several methods of obtaining adequate drought stress and fungal infection were developed to assess peanut genotypes resistance to aflatoxin contamination in breeding [2], [18].

The molecular mechanism of drought response has been extensively investigated in plants [15], [22] and many biochemical pathways and numerous genes proved to be involved. In the area of plant disease resistance, progress has been significant in understanding the molecular mechanisms of disease resistance in recent years [13], [29]. Although many advances have been made in understanding host resistance to Aspergillus infection and aflatoxin contamination [9], [17], little is known about the molecular mechanisms of drought stress-drought tolerance and resistance to A. flavus infection and afltoxin contamination. Because of the complexity of gene expression in plants under biotic and abiotic stresses, large scale analytic methods of gene expression should be applied. With the development of functional genomics, several techniques of gene expression analysis such as expressed sequenced tags (ESTs) and microarray have been used to study drought tolerance and disease resistance [12], [24], [40].

To prevent pre-harvest aflatoxin contamination, it will be necessary to have a more detailed understanding of the expression and function of the genetic material of peanut in response to the stresses and to develop specific gene probes for use in breeding resistant cultivars. The cDNA libraries for ESTs were constructed from immature pods of peanut line A13 (tolerant to drought stress and pre-harvest aflatoxin contamination) [28]. The objectives of this research were to identify the differentially expressed genes in response to A. parasiticus challenge under drought stress and to explore the mechanism of the resistance. In this paper, we report the identification and characterization of the expression patterns of the resistant genes or cDNAs in response to A. parasiticus infection and drought stress using microarray analysis and real-time PCR.