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
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.
Section snippets
Plant material and pathogen inoculation
A C. zedoaria accession (Accn. No. Cze512-11), resistant to P. aphanidermatum was used for isolation of the R-gene. In addition, four resistant C. zedoaria accessions (Cze516-04; Cze522-01; Cze527-13; Cze533-06), five susceptible C. zedoaria accessions (Cze102-03; Cze107-01; Cze112-09; Cze121-07; Cze123-02), a wild accession of Zingiber zerumbet resistant to P. aphanidermatum and a susceptible cultivated turmeric line C. longa cv. Surama were used for functional analysis. Four virulent strains
Isolation of full length cDNA of CzR1
In our attempt to clone a P. aphanidermatum responsive resistance gene from C. zedoaria, a pair of gene specific primer Czp11F and Czp11R were designed to amplify the pathogen responsive resistance gene candidate Czp11. Using RT-PCR, a single cDNA fragment of 538 bp was obtained. A forward gene specific primer 3PR1 designed according to the sequence information of the partial cDNA fragment and a 3′ RACE Adaptor primer was used for the amplification of 3′-end cDNA of CzR1, resulting in a single
Discussion
Rhizome rot, caused by P. aphanidermatum is the most devastating disease accounting for upto 60% of losses in turmeric productivity [2]. Control of rhizome rot in India and other countries relies extensively on fungicide infection. The obligatory asexual nature and availability of poor genetic information prevent the traditional crop breeding approaches for host resistance development in turmeric. C. zedoaria, a wild relative of turmeric has been lately identified as a source of resistance
Acknowledgments
The work is partially supported by grant from Department of Science and Technology, Govt. of India. BK gratefully acknowledges the financial assistance in the form of senior research fellowship from the Council of Scientific and Industrial Research, Government of India, New Delhi. We are thankful to the President, Siksha O Anusandhan University for his guidance and support.
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2016, Physiological and Molecular Plant PathologyCitation Excerpt :Another study reported the identification of a member of the pathogenesis related protein 5 (PR-5) from Z. Zerumbet that is expressed constitutively but upregulated in response to P. aphanidermatum [6]. In one of our previous study, we have isolated and characterized several nucleotide binding site-leucine rich repeats (NBS-LRRs) resistance gene analogs from different zingiberaceous species in response to P. aphanidermatum infection [7,8]. Transcriptional variability study of the resistance gene candidates revealed that CzP11, an R-gene analog showed higher expression in rhizome rot resistance lines of Curcuma zedoaria and C. longa [8].
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2014, Physiological and Molecular Plant PathologyCitation Excerpt :This suggests a prominent role of AsRGA29 either as a recognition molecule or as an accessory protein in the early steps of defense signaling towards FBR resistance in garlic. Similar expression pattern has been reported by Xa1 and Pib gene in rice [37,38], CsRGA23 in Cucumis [9] and CzR1 gene in Curcuma zedoaria [36]. There is enough evidence suggesting the critical role of signal molecules and phytohormones in regulating defense responses in plants [39,40].