Transcriptome analysis of phytophthora capsici tolerance in black pepper (piper nigrum L.)
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Date
2017
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Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara
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.
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