based on the following paper "Enhanced resistance to blast fungus in rice (Oryza sativa L.) by expressing the ribosome-inactivating protein alpha-momorcharin Qian Qian1, Lin Huang1, Rong Yi, Shuzhen Wang, Yi Ding∗ State Key Laboratory of Hybrid Rice, Department of Genetics, College of Life Sciences, Wuhan University, Wuhan 430072, Hubei Province, People’s Republic of China

1. 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

2. 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

3. Introduction
 Measures used against rice fugal disease
Spraying pesticides
RNA silencing
Over expression of
resistant gene

4. 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

5. RIP : Light of hope
RIP
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

6. 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

7. Methodology
Rice (Oryza sativa ssp.
japonica var. Nipponbare)
seeds,
Agrobac-terium
tumefaciens strain
EHA105
plasmid vector
pPRO
anti- -MC
polyclonal
antibody
Purified 28 kDa -MC
protein

8. 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

9. PCR and RT-PCR analysis
The cDNA
was used as a
template to
amplify and
further screen
transgenic
plants

10. Methodology
 Determination of the ˛-MC gene copy numbers
in the transgenic rice by Real-time PCR

11. 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
nitrocellulo
se
membrane
by semi-dry
trans-fer
The
membrane
was
incubated at
room
temperature
with 5%
(w/v)
defatted
milk in
TBST for 2
hour

12. Methodology
 Bioassay of transgenic rice plants
The concentration of properly
cultured and treated M. Grisea
fungal 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

13. 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

14. 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.

15. 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

16. 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

17. 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.

18. 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