Sains Malaysiana 47(10)(2018):
2291–2300
http://dx.doi.org/10.17576/jsm-2018-4710-05
The Effect of Pathogenesis-Related
10 (Pr-10) Gene on the Progression of Fusarium Wilt in
Musa acuminata cv. Berangan
(Kesan
Gen Berkaitan Patogenesis
10 (Pr-10) pada Perkembangan
Fusarium Layu dalam
Musa acuminata cv. Berangan)
NADIYA
AKMAL
BAHARUM1,2,
ROFINA
YASMIN
OTHMAN1,3,
YUSMIN
MOHD-YUSUF3,
BOON
CHIN
TAN3,
KAMILATULHUSNA
ZAID1I,3
& NORZULAANI KHALID1,3*
1Institute of Biological
Sciences, Faculty of Science, University of Malaya, 50603 Kuala
Lumpur, Federal Territory, Malaysia
2Department of Cell and
Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences,
Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor Darul Ehsan, Malaysia
3Centre for Research in
Biotechnology for Agriculture (CEBAR), University of Malaya, 50603
Kuala Lumpur, Federal Territory, Malaysia
4Centre for Foundation
Studies in Science, University of Malaya, 50603 Kuala Lumpur,
Federal Territory, Malaysia
Diserahkan: 15 Disember 2017/Diterima:
19 Jun 2018
ABSTRACT
PR-10 is a member of pathogenesis-related (PR)
genes elicited by the plant’s defense mechanism during pathogen
attack. Elevated expression of PR-10 upon different
pathogen invasions has been observed in many plant species suggesting
its role as an anti-bacterial, anti-viral and anti-fungal gene.
However, the effect of PR-10 in mitigating the infection of
Fusarium oxysporum f. sp. cubense
(Foc), the causal agent of Fusarium
wilt in banana has not been reported. In this study, the coding
sequences of PR-10 gene isolated from Foc resistant Musa acuminata
ssp. malaccensis (MaPR-10)
were integrated into a local Foc susceptible commercial banana cultivar, ‘Berangan’ via co-cultivation of embryogenic cell suspension
and Agrobacterium tumefaciens.
Out of 17 putative transgenic lines established, 11 of them positively
harbored MaPR-10. Among these, Line-19 plantlets showed the most
rapid in-vitro propagation and successfully over-expressed
the transgene. Following a nursery challenge experiment with a
virulent Foc race 4 (CI
HIR) isolate, about 30% of Line-19 plants showed
a one-week delay in disease progression when compared to the untransformed
controls. From the final evaluation performed in the 5th week-post-inoculation, the
leaf symptoms index (LSI) and rhizome discoloration index
(RDI)
of Line-19 was 3.4 and 6.1, respectively, indicating the disease
had progressed. The findings of this study enrich the current
existing knowledge on the roles of PR-10 in combating
fungal disease in plants.
Keywords: Fusarium oxysporum f. sp. cubense; Fusarium wilt; pathogenesis-related protein;
PR-10; transgenic banana
ABSTRAK
PR-10 merupakan antara gen berkaitan patogenan (PR)
yang dihasilkan oleh
sistem pertahanan tumbuhan semasa serangan patogen. Peningkatan ekspresi gen PR-10 ketika
ancaman beberapa
jenis patogen yang berbeza telah diperhatikan
di dalam banyak
spesies tumbuhan menunjukkan peranan gen ini sebagai agen
anti-bakteria, anti-virus dan
anti-kulat. Namun begitu, peranan
PR-10 dalam mengurangkan kesan jangkitan Fusarium oxysporum f.
sp. cubense (Foc), patogen penyebab penyakit layu pada
pokok pisang
masih belum pernah
dilaporkan. Dalam
kajian ini, jujukan
pengekodan PR-10 daripada Musa acuminata
ssp. malaccensis (MaPR-10)
telah diintegrasikan ke dalam genom
satu kultivar
pisang komersial tempatan, ‘Berangan’ melalui melalui kaedah penanaman antara Agrobacterium tumefaciens
dan ampaian
sel embriogenik. Daripada 17 jaluran pokok transgenik yang terhasil, 11 daripadanya disahkan positif mengandungi gen MaPR-10. Antara kesemua
jaluran yang terhasil,
Jaluran 19 menunjukkan kadar propagasi
in-vitro paling cepat dan
menunjukkan ekspresi
gen yang lebih tinggi
berbanding pokok kawalan yang tidak ditransform. Dalam penilaian tindak
balas Jaluran
19 terhadap jangkitan Foc ‘race’ 4 (C1 HIR), 30% daripada
anak pokok
menunjukkan penularan penyakit seminggu lebih lambat berbanding
pokok kawalan
yang tidak ditransform. Melalui penilaian terakhir pada minggu
ke-5 selepas inokulasi,
skor indeks sistem
daun (LSI) dan
indeks penyahwarnaan
rizom (RDI) bagi Jaluran 19 masing-masing ialah 3.4 dan 6.1, menunjukkan bahawa jangkitan Foc telah
merebak. Keputusan kajian uji kaji ini berupaya
meningkatkan maklumat
sedia ada berkaitan
peranan PR-10 dalam
menentang penyakit
kulat di dalam tumbuhan.
Kata kunci: Fusarium oxysporum f. sp. cubense; penyakit layu Fusarium; pisang transgenik; protein berkaitan patogenan; PR-10
RUJUKAN
Agarwal,
P. & Agarwal, P.K. 2014. Pathogenesis related-10 proteins
are small, structurally similar but with diverse role in stress
signaling. Molecular Biology Reports 41(2): 599-611.
Al-Obaidi, J.R., Mohd-Yusuf, Y., Chin-Chong,
T., Mhd-Noh, N. & Othman, R.Y. 2010. Identification
of a partial oil palm polygalacturonase-inhibiting
protein (EgPGIP) gene and its
expression during basal stem rot infection caused by Ganoderma
boninense. African Journal of
Biotechnology 9(46): 7788-7797.
Bezirganoglu,
I., Hwang, S.Y., Fang, T.J. & Shaw, J.F. 2013. Transgenic
lines of melon (Cucumis melo L. var. makuwa cv.‘Silver Light’) expressing antifungal
protein and chitinase genes exhibit
enhanced resistance to fungal pathogens. Plant Cell, Tissue
and Organ Culture 112(2): 227-237.
Bubner,
B. & Baldwin, I.T. 2004. Use of real-time
PCR for determining copy number and zygosity
in transgenic plants. Plant Cell Reports 23(5):
263-271.
Butler,
D. 2013. Fungus threatens top banana. Nature 504(7479):
195-196.
Chen,
Z.Y., Brown, R., Rajasekaran, K., Damann,
K. & Cleveland, T. 2006. Identification
of a maize kernel pathogenesis-related protein and evidence for
its involvement in resistance to Aspergillus flavus
infection and aflatoxin production. Phytopathology
96(1): 87-95.
Chen,
L., Zhong, H.Y., Kuang,
J.F., Li, J.G., Lu, W.J. & Chen, J.Y. 2011. Validation of
reference genes for RT-qPCR studies of gene expression in banana
fruit under different experimental conditions. Planta 234(2):
377-390.
Chin,
W.Y.W., Annuar, M.S.M., Tan, B.C. &
Khalid, N. 2014. Evaluation of a laboratory scale
conventional shake flask and a bioreactor on cell growth and regeneration
of banana cell suspension cultures. Scientia Horticulturae
172: 39-46.
Dodds,
P.N. & Rathjen, J.P. 2010. Plant
immunity: Towards an integrated view of plant-pathogen interactions.
Nature Reviews Genetics 11(8): 539-548.
García-Bastidas,
F., Ordóñez, N., Konkol,
J., Al-Qasim, M., Naser,
Z., Abdelwali, M. & Kema,
G.H.J. 2014. First report of Fusarium oxysporum
f. sp. cubense tropical
race 4 associated with Panama disease of banana outside Southeast
Asia. Plant Disease 98(5): 694-694.
Ghosh,
A., Ganapathi, T., Nath,
P. & Bapat, V. 2009. Establishment of embryogenic cell suspension cultures and Agrobacterium-mediated
transformation in an important Cavendish banana cv. Robusta (AAA).
Plant Cell, Tissue and Organ Culture 97(2): 131-139.
Go,
P.S. 2013. Expression of anti-toxoplasma scfv
antibodies in plants/Go Pei See. Master’s dissertation,
University of Malaya (Unpublished).
Jain,
S. 2008. In vitro mutagenesis in banana
(Musa spp.) improvement. Paper presented
at the International Conference on banana and plantain in Africa:
Harnessing international partnerships to increase research impact.
ISHS Acta Horticulturae
879: 605-614.
Kaeppler,
S.M., Kaeppler, H.F. & Rhee, Y.
2000. Epigenetic aspects of somaclonal variation
in plants. Plant Molecular Biology 43(2-3): 179-188.
Kim,
Y.J., Jang, M.G., Lee, H.J., Jang, G.H., Sukweenadhi,
J., Kwon, W.S. & Yang, D.C. 2014. Functional characterization of the pathogenesis-related protein family
10 gene, PgPR10- 4, from Panax
ginseng in response to environmental stresses. Plant
Cell, Tissue and Organ Culture 118(3): 531-543.
Kovács,
G., Sági, L., Jacon,
G., Arinaitwe, G., Busogoro, J.P., Thiry, E. & Remy, S. 2013. Expression
of a rice chitinase gene in transgenic
banana (‘Gros Michel’, AAA genome group)
confers resistance to black leaf streak disease. Transgenic
Research 22(1): 117-130.
Larkin,
P.J. & Scowcroft, W.R. 1981. Somaclonal variation - A novel source of variability from
cell cultures for plant improvement. Theoretical and Applied
Genetics 60(4): 197-214.
Lee,
O.R., Kim, Y.J., Balusamy, S.R.D., Khorolragchaa, A., Sathiyaraj, G.,
Kim, M.K. & Yang, D.C. 2012. Expression of
the ginseng PgPR10-1 in Arabidopsis confers resistance
against fungal and bacterial infection. Gene 506(1): 85-92.
Lin,
Y.H., Chang, J.Y., Liu, E.T., Chao, C.P., Huang, J.W. & Chang,
P.F.L. 2009. Development
of a molecular marker for specific detection of Fusarium oxysporum f. sp. cubense
race 4. European Journal of Plant Pathology 123(3):
353-365.
Mahdavi,
F., Sariah, M. & Maziah, M. 2012. Expression
of rice thaumatin-like protein gene
in transgenic banana plants enhances resistance to Fusarium
wilt. Applied Biochemistry and Biotechnology 166(4):
1008-1019.
Mak,
C., Mohamed, A., Liew, K., Ho, Y., Jain,
S. & Swennen, R. 2004. Early screening technique for Fusarium wilt resistance in banana micropropagated plants. Paper presented at the banana
improvement: Cellular, molecular biology, and induced mutations.
Proceedings of a meeting held in Leuven, Belgium, 24-28 September
2001.
Mao,
B., Liu, X., Hu, D. & Li, D. 2014. Co-expression of RCH10
and AGLU1 confers rice resistance to fungal sheath blight Rhizoctonia
solani and blast Magnorpathe
oryzae and reveals impact on seed
germination. World Journal of Microbiology and Biotechnology
30(4): 1229-1238.
Maziah, M., Sariah, M. &
Sreeramanan, S. 2007. Transgenic banana
Rastali (AAB) with β-1, 3-glucanase
gene for tolerance to Fusarium wilt race 1 disease
via Agrobacterium-mediated transformation system. The
Plant Pathology Journal 6: 271-282.
Moiseyev, G.P.,
Beintema, J.J., Fedoreyeva,
L.I. & Yakovlev, G.I. 1994. High sequence similarity between a ribonuclease from
ginseng calluses and fungus-elicited proteins from parsley indicates
that intracellular pathogenesis-related proteins are ribonucleases.
Planta 193(3): 470-472.
Negi,
S., Tak, H. & Ganapathi,
T. 2014.
Cloning and functional characterization of MusaVND1
using transgenic banana plants. Transgenic Research
24(3): 571-585.
Ordonez,
N., García-Bastidas, F., Laghari,
H.B., Akkary, M.Y., Harfouche,
E.N., Al Awar, B.N. & Kema,
G.H.J. 2016.
First report of Fusarium oxysporum
f. sp. cubense tropical
race 4 causing Panama disease in Cavendish bananas in Pakistan
and Lebanon. Plant Disease 100(1): 209.
Pérez-Vicente,
L., Dita, M.A. & Martínez-de
la Parte, E. 2014.
Technical manual: Prevention and diagnostic of Fusarium wilt
(Panama disease of banana caused by Fusarium oxysporum
f. sp. cubense tropical
race 4 (TR4). www.fao.org website: http:// www.fao.org/fileadmin/templates/agphome/documents/
Pests_Pesticides/caribbeantr4/13ManualFusarium.pdf.
Accessed on 26 June 2015.
Ploetz, R.C.
2015.
Management of Fusarium wilt of banana: A review with special
reference to tropical race 4. Crop Protection 73: 7-15.
Shekhawat, U.K.S.
& Ganapathi, T.R. 2014. Transgenic banana plants
overexpressing MusabZIP53 display severe growth retardation
with enhanced sucrose and polyphenol oxidase activity. Plant
Cell, Tissue and Organ Culture 116(3): 387- 402.
Soh, H.C., Park, A.R., Park,
S., Back, K., Yoon, J.B., Park, H.G. & Kim, Y.S. 2012. Comparative analysis of pathogenesis-related protein 10 (PR10)
genes between fungal resistant and susceptible peppers.
European Journal of Plant Pathology 132(1): 37-48.
Sreedharan, S.,
Shekhawat, U.K. & Ganapathi,
T.R. 2013.
Transgenic banana plants overexpressing a native plasma membrane
aquaporin MusaPIP1;2 display high tolerance levels to different abiotic stresses.
Plant Biotechnology Journal 11(8): 942-952.
Sreedharan, S.,
Shekhawat, U.K.S. & Ganapathi,
T.R. 2012.
MusaSAP1, a A20/AN1 zinc finger
gene from banana functions as a positive regulator in different
stress responses. Plant Molecular Biology 80(4-5): 503-517.
Sreeramanan, S.,
Maziah, M., Rosli,
N., Sariah, M. & Xavier, R. 2006. Particle bombardment-mediated
co-transformation of chitinase and β-1,
3 glucanase genes in banana.
Biotechnology 5(2): 203-216.
Swarupa, V.,
Ravishankar, K. & Rekha,
A. 2014.
Plant defense response against Fusarium oxysporum and strategies to develop tolerant genotypes
in banana. Planta 239(4): 735- 751.
Vishnevetsky, J.,
White, Jr. T.L., Palmateer, A.J., Flaishman, M., Cohen, Y., Elad,
Y. & Dgani, O. 2011. Improved
tolerance toward fungal diseases in transgenic Cavendish banana
(Musa spp. AAA group) cv. Grand Nain. Transgenic
Research 20(1): 61-72.
Zhang,
G., Li, Y.M., Zhang, Y., Dong, Y.L., Wang, X.J., Wei, G.R. &
Kang, Z.S. 2010.
Cloning and characterization of a pathogenesis-related
protein gene TaPR10 from wheat induced by stripe rust pathogen.
Agricultural Sciences in China 9(4): 549-556.
*Pengarang untuk surat-menyurat; email: lani@um.edu.my