Transcriptome analysis of phytophthora capsici tolerance in black pepper (piper nigrum L.)

Abstract

Black pepper (Piper nigrum L.) regarded as the ‘king of spices’ is one of the most important spices in the world, cultivated in more than 30 countries. Foot rot disease caused by the fungal pathogen Phytophthora capsici Leonian is the major production constraint since 1951. Tropical climates favour the development of foot rot disease wherein the survival of pathogen is exceptionally high, leading to complete crop losses. The long term survivability of oospores in soil, a wide host range, long-distance movement of the pathogen in surface water, presence of fungicide-resistant pathogen population, and a lack of resistant high yielding cultivars make this the most deadly disease. Understanding of molecular basis of Phytophthora resistance can help control the disease by gene stacking, molecular breeding and exploiting the natural resistance prevailing in wild species of black pepper. The study entitled “Transcriptome analysis of Phytophthora capsici tolerance in black pepper (Piper nigrum L.)” was carried out with the objective to understand the differential expression of genes during Phytophthora infection in susceptible and tolerant black pepper cultivars through DDRT-PCR analysis on mRNA. IISR Subhakara, the susceptible cultivar along with IISR Shakthi, the tolerant cultivar were used for the evaluation of transcriptome profiles in response to the infection. Phytophthora capsici pure culture has been isolated and maintained on Carrot Agar Medium (CAM). Artificial inoculation with mycelial discs (5 mm) was done on the tender leaves of two black pepper cultivars and total RNA was isolated at 0, 2, 4, 6, 12, 24 and 48 hours after inoculation (hai) using TRIzol reagent. The RNA was quantified using NanoDrop® spectrophotometer and electrophoresed on 1 per cent formaldehyde agarose denaturing gel. cDNA first strand was synthesized from the total RNA using RevertAid H minus first strand synthesis kit and second strand was synthesized using anchored and arbitrary primers (DDRT-PCR). The DDRT-PCR product was electrophoresed on 6 per cent urea polyacrylamide sequencing gel. The transcriptome profiles were distinct for each of the primer combinations in tolerant and susceptible cultivars. A total of twelve differentially expressed bands were identified, of which two were upregulated and one was down-regulated in tolerant cultivar. The bands are eluted and amplified selectively using the same primer combination. The selective-PCR product was sequenced and the sequences were analysed in-silico for characterisation of fragments and functional annotation of genes and metabolic pathways. Sequence of the differentially expressed band (455 bp) A2 generated with the primer combination AP1-T11C was similar to that of Premnaspirodiene oxygenase gene-like, which is involved in the biosynthesis of solavetivone and piceatannol, potent antifungal phytoalexins, which inhibits the spore germination and germ tube growth. Sequence of band A2 (455 bp) generated with the primer combination AP1-T11C was similar to Cytochrome P450 gene is involved in detoxification pathway which detoxifies the fungal protein elicitors and native toxins. Sequence of band J2 (405 bp) generated with the primer combination AP6-T11C was similar to Phosphoserine phosphatase. This super family of proteins stands for the activation of defense related genes in the nucleus through extra chromosomal transcription activation, reactive oxygen species (ROS) regulation and signaling pathways by the generation of nuclear localization signals (NLS) and it acts through ABA mediated, ethylene mediated or cytokine mediated signaling pathways. Similarly, BLASTp analysis showed similarity with UPL5 from M4 sequence (177 bp) and ANKHD1, MAPK alpha 1, protein kinase superfamily and GPCR from M1 sequence (174 bp). GPCR (G protein coupled protein receptor) cleaves the signal peptides thereby activating the protein molecules. G3BP1 (GTPase activating protein binding protein) is stress- granule nucleating protein which stabilizes the RNA transcript and helps in the formation of RNA granule mainly against viruses. ANKHD1 is an anti-apoptotic regulating domain which protects the normal cell from degradation. UPL5 (Ubiquitin protein ligase 5) is involved in 26S proteasome mediated protein degradation whereas the rRNA genes induces non host pathogen derived hypersensitivity reactions (HR). The fragments which are differentially expressed, upregulated and downregulated in tolerant cultivar during the time of pathogenesis implies the resistance mechanism by which the black pepper tackles Phytophthora capsici attack. The sequence information could be further used to design SCAR primers which would be useful in distinguishing tolerant and susceptible cultivar. It can also enrich the genetic base by incorporation of identified genes through genetic transformation for disease tolerance.