Identification and molecular characterisation of vascular-related defense genes against papaya dieback disease
Malaysia is one of the world’s main exporters of papaya with an export value worth RM120 million per year. An outbreak of papaya dieback disease on 2003 lowered the market value up to 60% from the previous year, and remains affected since. Erwinia mallotivora, the causal bacteria for papaya di...
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| Format: | Thesis |
| Language: | English |
| Published: |
2017
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/69535/1/fbsb%202017%2026%20ir.pdf |
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| Summary: | Malaysia is one of the world’s main exporters of papaya with an export value worth
RM120 million per year. An outbreak of papaya dieback disease on 2003 lowered the
market value up to 60% from the previous year, and remains affected since. Erwinia
mallotivora, the causal bacteria for papaya dieback disease, enters the papaya plant
either through natural openings or wounds, to penetrate into the petioles and stem, and
subsequently colonizes the entire vascular system. This results in the appearance of
water-soak lesion at the infected region and disruption of the plant’s upper meristem
region, which produces papaya fruits. To date, the only solution to this problem is by
demolishing all the infected papaya plants as it can easily be transmitted to other plants
nearby. An alternative approach such as genetic engineering to avoid mass destruction
of matured papaya plants is deemed critical. Hence, the aims of this study were to
identify potential vascular-related defense genes using bioinformatics approach, isolate
and characterize the genes from Eksotika papaya and subsequently to assess the
functionality of the genes in transformed papaya seedlings challenged with E.
mallotivora. This study has successfully identified vascular-related defense genes
against papaya dieback disease using bioinformatics approach. After data mining of
bioinformatics databases including The Arabidopsis Information Resources (TAIR),
Arabidopsis thaliana Trans-factor and cis-element Prediction Database (ATTED),
Phytozome, and National Center for Biotechnology Information (NCBI), identified five
potential genes were then mapped onto the Kyoto Encyclopedia of Genes and Genomes
(KEGG) pathway of Arabidopsis model plant. Orthologs of the Glycerol kinase
(NHO1), Pathogenesis-related protein 1 (PR1b), Leucine-rich repeat receptor-like
serine/threonine-protein kinase (EFR), RPM1 interacting protein 4 (RIN4) and
Mitogen-activated protein kinase 4 (MPK4) genes were isolated and fully characterized
from Carica papaya var. Eksotika I. NHO1 gene with an estimated size of 1572 base
pair (bp) was hypothesized as defense-related gene against pathogen. The similar role
was predicted with PR1b gene (492 bp), while EFR gene (741 bp) was postulated to be
involved in pathogen signaling. In addition, RIN4 gene (540 bp) was suggested to be a
member of resistance inducer upon pathogen invasion. MPK4 gene (1125 bp) was
estimated to work in a cascade of mediating various responses against pathogens
through different signaling pathway of defense responses. These genes were then sub cloned, in a sense orientation, into the pEAQ virus vector for subsequent
transformation into papaya seedlings via Agrobacterium tumefaciens strain GV3101.
The constructs were designated as pEAQ.NHO1, pEAQ.PR1, pEAQ.EFR, pEAQ.RIN4
and pEAQ.MPK4. Leaves of two months old papaya seedlings were infiltrated with A.
tumefaciens strain GV3101 (OD600 = 1.5) harbouring each respective constructs. The
transformed papaya seedlings were then challenged with E. mallotivora (cfu = 106) and
exhibited disease symptoms development as early as day two after infection. Further
profiling of the transgene expression in the papaya seedlings via real-time polymerase
chain reaction (qRTPCR) analysis resulted in functional expression against the causal
bacteria of this papaya dieback disease. Overexpression of PR1b and NHO1 genes
resulted in high expression level in infected plants compared to the uninfected plants.
While overexpression of MPK4 and RIN4 showed high fold changes compared to
control plants, expression of MPK4 exhibited a repressed defense response via earlier
development of symptoms in both infected and uninfected plants. In addition,
overexpression of EFR showed a down-regulation expression for both infected and
uninfected plants. These findings will lay the foundation for subsequent studies in
developing a conceivable solution against this papaya dieback disease. |
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