enhanced resistance to blast fungus in rice (
TRANSCRIPT
Enhanced resistance to blast
fungus in rice (Oryza sativa
L.) by expressing the
ribosome-inactivating protein
alpha-momorcharin
A presentation by :
Md. Rezwan Ul Haque
Reg no: 2012421024
Introduction
Rice blast is a ubiquitous problem, found in more than fifty-eight countries around the world
Rice blast caused by Magnaporthe grisea is one of the three major diseases that seriously affect the rice production
Rice blast is characterized by the appearance of lesions on the leaves, nodes, and panicles
Introduction Measures used against rice fugal disease
Spraying pesticides
RNA silencing
Over expression of resistant gene
Introduction:
Achievement so far
• Since the genome of japonica rice was sequenced, more and more resistant genes have been located
• Before June 2012, at least 63 loci involved in resistance blast fungus, containing 77 major genes, were found in rice genome
• Some of the genes identified had a certain degree of antifungal activity in bioassay, e.g., Pid3-A4
RIP : Light of hopeR
IP
Ribosome-inactivating proteins (RIPs) act as an N-glucosidase by removal of one or more adenine residues from 28S rRNA to inhibit protein synthesis
This property of RIPs may result in cell death, and thus inhibit the degree of pathogenesis during bacterial
infection
Expression of curcin2 in transgenic tobacco plants clearly demonstrated antifungal activity
GOAL OF THE STUDY
. In this study, it is found that ˛-MC gene encoding a type 1 RIPs in rice by using a transgenic method. These transgenic rice plants clearly show an enhanced the resistance to rice blast.
Alpha-momorcharin( -MC),
Belonging to type 1
RIPs, has a clean effect
on resistance to fungi
in addition to its inherent N-glycosidase
activity and DNA-
nuclease activity
Methodology
Rice (Oryza sativa ssp. japonica var. Nipponbare)
seeds,
Agrobac-teriumtumefaciens strain
EHA105
plasmid vector pPRO
Purified 28 kDa -MC protein
anti- -MC polyclonal antibody
Methodology
Construction and transformation of plant
expression vector
The 2 × 35S promoter fragment and ˛-MC were amplified by PCR and then cloned into the pMD18-T vector
The recombinant T-vectors were digested by and the fragments with cohesive end were cloned into the pPRO plasmid in the proper orientation. The recombinant plas-mid (2 × 35Sp- -MC-nos) was transformed into the agrobacterium strain EHA105
The recombinant plant vector was transformed into Nipponbare calli using the A. tumefaciens mediated method in N6
medium
PCR and RT-PCR analysis
The cDNAwas used as a template to amplify and
further screen transgenic
plants
Methodology
Determination of the ˛-MC gene copy numbers
in the transgenic rice by Real-time PCR
Methodology
Western blot analysis
proteins were extracted from 0.2 g of young leaves from each transgenic rice plants by trituration in liquid-N2
and homog-enized with 1 ml extraction buffer
The prepared proteins were separated by 12% SDS-PAGE and transferred onto a nitrocellulose membrane by semi-dry trans-fer
The membrane was incubated at room temperature with 5% (w/v) defatted milk in TBST for 2 hour
Methodology
Bioassay of transgenic rice plants
The concentration of properly cultured and treated M. Griseafungal spore was kept approximately 1 × 105 spores/ml
When rice seedlings (T1 lines) and wild type (WT) had four -to five -leaf stage in a plant growth chamber under specific condition, each leaf was sprayed with 250 l of the M. grisea spores suspension
Ten days after inoculation, the severity of the rice blast infection was evaluated using the detection and identification criteria set by IRRI
RESULTS:
Production of transgenic rice plants
The plant expression vector
pCAMBIA1301-pPRO (Fig. 1) containing
the ˛-MC gene under the control of the 2
× 35S promoter was used and
transferred to Nipponbare (Japonica) rice
via Agrobacterium tumefaciens-mediated
method.
Transgenic rice plants were screened on
N6 medium containing hygromycin (50
mg/l) for four weeks
. After three or more weeks, a total of
eighteen transgenic plantlets were
regenerated and grown in the
greenhouse
Results:
Expression of the ˛-MC gene in transgenic rice plants
To examine the presence and expression of the ˛-MC gene, all independent transgenic plant seedlings (T0 generation) were sub-jected to PCR and RT-PCR using -MC specific primers.
As shown in Fig. 1. Lanes 2, 3, 4, 5, 7, 8, 11, 14 and 15 in Fig. 1A, correspond to Lanes B1, B2, B3, B4, B5, B6, B7, B8 and B9 in Fig. 1B–D.
These results show that the ˛-MC gene was successfully inserted into the rice genome in these nine independent transgenic plants, while the control (lane 18, wild type) did not contain the 861 bp band in the corresponding position.
Results:
Expression of the ˛-MC gene in transgenic rice plants
The qRT-PCR data showed that
the expression level of -MC
exhibited different among
different transgenic lines.
However, there is no correlation
between resistance and copy
number
According to the instruction
of fluorescent quantitative
PCR, the correlation
coefficients of standard
curves should meet the
condition R2 > 0.98
Results:
Expression of the ˛-MC gene in transgenic rice plants
The -MC protein obtained from
leaves of four identified T0
transgenic lines was further
proved by western blot analysis
Anti- -MC polyclonal antibodies
used against purified -MC
protein (28 kDa), which was
expressed in a prokaryotic
expression system, were first
tested by western blot
anti- -MC polyclonal antibodies could
also hybridize with the -MC protein from
transgenic rice plants and it was
approximately 38 kDa
Disease resistance analysis of the transgenic
plants
After spraying a spore
suspension on the leaves of T1
and WT generation seedlings,
for ten days each leaf of the
transgenic and WT control
plants was evaluated
As the infection time passed, some disease
spots were observed to appear on the leaves
and did not affect the growth of plants, but the
damage on the control plants became more
serious with time
Disease level Number of leaves
B2a B4a B7a B9a WTb
0 3 8 12 28 11
1 1 2 2 8 3
2 0 1 2 1 5
3 0 0 0 0 2
4 0 0 0 0 5
5 0 1 1 0 4
a Transgenic rice plants(T1).
b Control rice plants.
Conclusion
According to the criteria of International Rice Research Institute
standard, the mean values for morbidity and disease index numbers
were 29.8% and 14.9%, respectively, which were lower than for WT.
It is unclear whether RIPs could impact plant fitness and however our
results suggest that the -MC protein is an effective antifungal protein
preventing rice blast in transgenic rice
In conclusion, this study shows that rice blast resistance is enhanced
in transgenic rice plants by the expression of the - MC protein