Molecular characterization and functional analysis of CzR1, a coiled-coil-nucleotide-binding-site-leucine-rich repeat R-gene from Curcuma zedoaria Loeb. that confers resistance to Pythium aphanidermatum

Highlights

• CzR1, a major R-gene was isolated from Curcuma zedoaria using RACE-PCR strategy.

• Structural analyses classified CzR1 as a non-TIR-CC-NBS-LRR R-gene.

• RT-PCR revealed specific expression of CzR1 only in pathogen resistant plant types.

• CzR1 shows upregulation in response to different strains of Pythium aphanidermatum.

• CzR1 shows highest transcript accumulation in the rhizome tissues of C. zedoaria.

Abstract

Rhizome rot disease caused by necrotrophic oomycete Pythium aphanidermatum is responsible for upto 60% of yield losses in turmeric (Curcuma longa L). However, Curcuma zedoaria L, a wild relative of turmeric, is resistant to P. aphanidermatum and has been proposed as a potential donor for rot resistance to C. longa. We used a previously isolated resistance gene candidate Czp11 from C. zedoaria as a template to characterize a major resistance gene CzR1 through candidate gene approach in combination with RACE-PCR strategy. CzR1 encodes a 906 amino acid predicted protein with a calculated pI of 8.55. Structural and phylogenetic analyses grouped CzR1 within the non-TIR (homology to Toll/interleukin-1 receptors) subclass of NBS-LRR R-genes. Reverse transcription PCR revealed specific transcript expression of CzR1 only in P. aphanidermatum resistant lines of C. zedoaria and Zingiber zerumbet, another resistant wild species of the family Zingiberaceae. Semi quantitative RT-PCR analysis showed constitutive expression of CzR1 which gets significantly upregulated in response to infection by different strains of P. aphanidermatum. Although, the expression of CzR1 was reported in the root, leaf and rhizome tissues of C. zedoaria, the relative transcript expression was highest in the rhizomes. Elucidation of these molecular characteristics of CzR1 will pave way towards a broad spectrum rhizome rot resistance development in the cultivated turmeric.

Introduction

Turmeric (Curcuma longa Loeb; Zingiberaceae) is one of the most important herb in the tropical and sub-tropical countries. Its rhizome is used as a spice, food preservative, coloring agent, and in the traditional systems of medicine [1]. Recent utility of turmeric by the pharmaceutical industries as a source of antioxidant, hepatoprotectant, anti-inflammatory in addition to its use in cardiovascular and gastrointestinal disorders has categorized it as a major industrially important crop of high demand [1]. The International Trade Centre, Geneva, has estimated an annual growth rate of 10% in world demand for turmeric [1]. However, crop losses upto 60% has been realized in the recent times mainly due to the infection by a necrotrophic oomycetic fungus Pythium aphanidermatum causing the rhizome rot disease in turmeric [2]. Utilization of chemical pesticide for the control of rhizome rot is highly unsatisfactory and growing cultivars with inherent resistance to P. aphanidermatum can be the most cost-effective and environment friendly method of protecting turmeric plants. However, the obligatory asexual nature and high stigmatic incompatibility of the extant turmeric lines prevents the establishment of a conventional breeding approach. A genetic transformation approach using foreign genes could be the only solution towards development of rhizome rot resistance in turmeric. Although the transformation technology in turmeric is ready [3], no resistance genes have been cloned and transferred to susceptible turmeric cultivars against the most destructive turmeric diseases.

Utilization of the plant disease resistance genes (R-genes) has been the major means towards detection of the pathogen effectors and activation of the plant defense response. R gene-mediated recognition of specific pathogen virulence factors as invasion signals results in the activation of a series of rapid cellular defense signaling often leading to swift local cell death at the infection site through hypersensitive response (HR) [4]. Around 70 different plant R genes grouped into five major classes have been isolated and characterized from different plant species during the last 15 years for resistance to a wide spectrum of pathogens, including bacteria, viruses and fungi [5]. Among them, the largest class of R-gene encodes proteins that have a putative amino-terminal signaling domain, a nucleotide binding site (NBS) and a series of carboxy-terminal leucine rich repeats (LRRs) [6]. These genes have been classified into the TIR subclass and the nonTIR subclass on the basis of the presence/absence of an N-terminal Toll/interleukin receptor (TIR) domain [7]. Genes in the TIR group are known among both monocotyledonous and dicotyledonous species while the non-TIR group typically includes a coiled-coil (CC) sequence or putative leucine zipper (LZ) at the N terminus among the monocots [8]. The LZ domain is believed to facilitate the formation of CC structure to promote oligodimerization with a wide variety of proteins although its actual task in R-gene function is still unknown [9]. The NBS region is thought to regulate signal transduction through nucleotide triphosphate (NTP) hydrolysis and conformational changes [10], [11]. The LRRs are the major sites for protein–protein interaction and determines the specificity for the pathogen avirulence factor(s) [12].

Our laboratory has been engaged in characterizing resistance related sequences in turmeric against P. aphanidermatum through candidate gene approach. The genetic variation for disease resistance is poorly developed in the cultivated turmeric [13]. Our earlier attempt to clone resistance related sequences from wild turmeric genotypes resulted in the isolation of expressive resistance gene candidates (RGCs) from Curcuma aromatica, Curcuma angustifolia and Curcuma zedoaria [14]. Interestingly, the expression of Czp11 RGC from C. zedoaria was uniquely found associated only with P. aphanidermatum resistant lines. This is in accordance to the earlier report that C. zedoaria L, a wild relative of turmeric show exclusive resistant against P. aphanidermatum [15]. In the present report, we have cloned and characterized a P. aphanidermatum responsive NBS-LRR R-gene CzR1 in C. zedoaria using Czp11 RGC as the reference, analyzed its phylogeny, expression pattern and discussed the possible function of the R-gene encoded protein in regulating defense mechanism in C. zedoaria.