Transcriptome based identification of genes imparting bacterial leaf blight (Xanthomonas oryzae) resistance in rice (Oryza sativa L.)
Loading...
Date
2017
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
AAU, Anand
Abstract
Rice is one of the most important staple foods for a larger part of
the world’s population. It is a rich source of carbohydrates, energy,
thiamine, pantothenic acid and folic acid. Because world-wide rice
production has been severely affected by one of the major disease
referred to as bacterial leaf blight caused by Xanthomonas oryzae pv.
oryzae (Xoo), early efforts has been focusing on cloning and utilizing of
disease resistance (R) genes in rice for resistance breeding.
In the present study, thirty day old seedlings of BB resistant (IR
64) and susceptible (GR 11) genotypes were inoculated with and
without BB (4 x l04 cells /ml). The seedlings were harvested 36 hours
post inoculation for transcriptome sequencing employing the Miseq
Illumina NGS sequencer after assaying the normal range for quantity
and quality of total RNA, mRNA and cDNA.
Transcriptome sequencing yielded 19311304 bp data with an
average read length of 150 bp and constituted of 18440773 high
quality reads which were assembled employing Trinity assembler for
functional annotation. Trinity assembler gave assembly matrix (N50 of
2,453), proteome coverage and read alignment (90%) resulting in
30,000 contigs and 2,35,433 singletons/transcripts which were
subjected to gene ontology (GO) following functional annotation.
ii
The maximum number of gene ontology was ascribed to cellular
components, molecular function and biological processes showed that
the maximum transcripts belonged to plastid, mitochondria and
plasma membrane; nucleotide binding, kinase activity, DNA binding;
cellular protein modification response to abiotic stress and biotic
stress, respectively. Rice transcripts showed highest homology to
Setaria italica and maximum blast hits with Hordeum vulgare. The
maximum number of annotated transcripts was ascribed to
transferase, hydrolase and oxidoreductase class of enzymes. The
highly represented transcripts included disease resistance genes,
pathogenesis related proteins, transcription factors (MYB, WRKY),
signaling molecules (MAPK), enzymes of oxidative burst and LRR
domain. These proteins were assumed to be involved in signaling
networks induced in rice during BB infection. The transcripts were
further subjected to confer their metabolic involvement through
biochemical pathway through Kyoto encyclopedia of genes and
genomes.
Total 118 metabolic pathways were identified in rice
transcriptome. Metabolic pathway enrichment ascribed the maximum
number of transcripts in Glutathione metabolism leading to activation
of defence responses called systemic acquired resistance (SAR).
Metabolic pathway for purine metabolism, phenylalanine metabolism
and starch and sucrose metabolism were prominently present.
Transcriptomes of resistant and susceptible rice genotypes (with
and without inoculum) were further screened for differential gene
expression analysis by mapping individual reads to the assembly.
Among the resistant and susceptible genotypes (with and without
inoculum), 1314 transcripts were either up regulated or down
regulated. The number of differentially expressed transcripts between
the samples resistant inoculated and susceptible inoculated was 154.
Further confirmation and validation was carried out through
quantitative real time PCR.
iii
Relative quantification of defence related transcripts by
quantitative real time PCR was performed using a-tubulin as
endogeneous control. The relative expression of defence related genes
associated with BB disease MYB, bZIP; defence related enzymes
including lipoxygenase (LOX), phenylalanine ammonia lyase (PAL),
respiratory burst oxidase (RBO), beta glucanase (BGLUC),
Ribonuclease (RNase), Endo chitinase (CHI), signaling molecules,
MAPK and CDPK showed up regulation in resistant inoculated than
susceptible inoculated.
Overall results of transcriptome study generated the information
on involvement of MYB, WRKY, mitogen activated protein kinases,
cellular ATPases, calcium dependent protein kinase and
phenylalanine ammonia lyase transcription factors and the genes
associated with resistance involved R genes homologous with the cell
wall degrading enzymes like chitinase, 3-1,3 glucanases and metabolic
pathway coding enymes polygalacturonase inhibiting proteins (PGIPs),
cysteine proteases in the process of BB resistance.
It could be inferred that defence responses associated with BB
in rice were activated by hypersensitive response arising due to gene
to gene interaction and systemic acquired resistance associated with
pathogenesis related proteins, transcription factors, signaling
molecules and enzyme related to glutathione pathway
Description
Keywords
agriculture, biotechnology, identification