Characterization of novel oil palm defense-related transcription factors during biotrophic and necrotrophic infection phases of Ganoderma boninense
Basal stem rot (BSR) disease, caused by Ganoderma boninense has been pinpointed to be one of the major factors that contribute to the decline in yield of oil palm. Hemibiotroph pathogen such as G. boninense manipulates the host defense mechanisms to strategically infect host plant by switching fr...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2020
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/90886/1/IPTSM%202020%204%20-%20IR.pdf |
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| Summary: | Basal stem rot (BSR) disease, caused by Ganoderma boninense has been pinpointed to
be one of the major factors that contribute to the decline in yield of oil palm.
Hemibiotroph pathogen such as G. boninense manipulates the host defense mechanisms
to strategically infect host plant by switching from biotrophic to necrotrophic phase.
Recognizing the early infection phase of G. boninense by identifying phase-specific oil
palm transcription factors (TFs) may offer opportunities to tackle or attenuate the
progress of infection. The fragmentary information on molecular interactions between
plant and hemibiotroph should be explicated to identify the key molecular mechanisms
of pathogenesis and plant immunity. Thus, this study is an attempt to recognize specific
TFs as ‘key’ biomarkers in identifying oil palm defense mechanisms during biotrophic
and necrotrophic infection phases of G. boninense. Artificial infection of G. boninense
on oil palm was performed by using Ganoderma-inoculated rubber wood blocks (RWBs)
and bare RWBs serves as mock-treatment presenting abiotic stress. In order to identify
oil palm defense response during early interaction with G. boninense, transcriptomic
analysis of root tissues at different time points of 3, 7 and 11 days post inoculation (d.p.i)
was carried out. High-throughput RNA-seq data analysis has revealed two
distinguishable expression profiles of oil palm genes that formed the basis for deducing
biotrophic phase at early interaction (3 d.p.i) which switched to initiation of necrotrophic
phase at later stage (11 d.p.i) of infection. Based on the findings, the present study
focused on identifying differentially expressed genes (DEGs) encoding TFs from the
generated RNA-seq data. A total of 106 upregulated and 108 downregulated DEGs of
TFs were identified. There are four established defense-related pathways that have been
presented whereby reported genes involved in cell wall modification, reactive oxygen
species (ROS)-mediated signaling, programmed cell death (PCD) and plant innate
immunity were differentially expressed. The genes were found to be either upregulated
or downregulated during the two distinct infection phases. Multiplex semi-quantitative
RT-PCR was conducted to screen for defense-related TFs independent of abiotic stress.
Normalized band intensity of Ganoderma-treated (GT) samples were compared to the
mock-treated (MT) samples to estimate the mRNA expression level between groups. The
expression patterns of eight candidate TFs genes including EgJUB1, EgERF113, EgTCP15, EgNAC29, EgEIN3, EgMYC2, EgNAC83 and EgMYB122 were further
quantified via quantitative Real-Time PCR (qPCR). Both EgJUB1 and EgERF113 were
found to be specifically upregulated under biotic stress at all time points. The findings
discovered upregulation of EgJUB1 during biotrophic phase while EgERF113
demonstrated prominent upregulation as oil palm switches to defense against
necrotrophic phase. Characterization of EgJUB1 and EgERF113 was performed via in
vivo Yeast One-Hybrid (Y1H) assay and in vitro electrophoretic mobility shift assay
(EMSA). JUB1 has been reported to bind to NAC binding site (NACBS) motif during
abiotic stress. The present study is the first report on the binding activity of EgJUB1 to
secondary wall NAC binding element 1 (SNBE1) which was present in the promoter
region of EgHSFC-2b having similar expression profile as EgJUB1. SNBE1 motifs with
single nucleotide change at either 5th or 18th position have been found in the promoter
regions of a few TFs that co-expressed with EgJUB1, including EgHSFB-4b and
EgGAMYB X2. Meanwhile EgERF113 binds to GCC-box and DRE/CRT motifs
promoting plasticity in upregulating downstream defense-related genes against G.
boninense attack. Sequence analysis revealed the presence of NAC DNA binding domain
(DBD) in EgJUB1. Amino acid change from phenylalanine (F) to tryptophan (W) at 14th
position of EgERF113 DBD proved the binding specificity to both GCC-box and
DRE/CRT motifs. Prolonged treatment revealed oil palm seedlings succumbing to the
G. boninense infection. Mature basidiomata of G. boninense was observed at 24 weeks
post inoculation (w.p.i) and the infection culminated in plant death. Overall, our findings
propose EgJUB1 and EgERF113 as key TFs in orchestrating the oil palm defense
mechanisms during biotrophic and necrotrophic infection phases of hemibiotrophic G.
boninense, respectively. |
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