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