CLONING AND EXPRESSION ANALYSIS OF RICE GENES

INVOLVED IN INFECTION WITH HIRSCHMANNIELLA ORYZAE REGMI, HOMAN (S1, ENTOMOLOGY)

Promoter

Prof. Dr Godelieve Gheysen

Faculty of Bio-Science Engineering

Gent University, Belgium

Supervisors

Dr Tina Kyndt and Lander Bauters

Faculty of Bio-Science Engineering

Gent University, Belgium

INTRODUCTION

Rice

major staple food of nearly half of the world’s population

model monocot plant

Genome size: 430 Mb

around 57 thousands sequenced rice genes available in rice genome database for free

Hirschmaniella oryzae

migratory endo-parasite

well adapted in deep water (anaerobic condition)

irrigated rice covers about 72% of total rice production area

the most prevailing nematode in rice world wide

Rice nematode molecular interaction

defense response genes believed to be involved in the

resistance mechanism

very less work done to know about rice and H. oryzae

molecular interaction

RICE GENES SELECTED FOR THE STUDY Locus Gene Expression under H.

oryzae infection

(Kyndt, unpublished)

Other information

LOC_Os08g09080 Cupin domain

containing protein

Up-regulated upregulated upon blast (Swarbrick et

al.,2008) and Striga infection (Marcel

et al., 2010)

LOC_Os11g44910 DEAD-box ATP-

dependent RNA

helicase

Down-regulated down-regulated at early phase of rice

germination (Huang et al., 2009)

LOC_Os03g08880 purine permease Up-regulated upregulated upon M. oryze infection

(Marcel et al., 2010)

LOC_Os04g33830 membrane protein Down-regulated down-regualted in resistant Nipponbare

under Striga hermonthica infection

(Swarbrick et al.,2008)

LOC_Os04g50100 RING-H2 finger

protein

Down-regulated ATL5G has been suggested to be

involved in the early steps of the plant

defense signaling pathway

LOC_Os02g39360 B-box zinc finger

family protein

Down-regulated down-regulated under Xanthomonas

oryzae pv. oryzicola infection (Zhou et

al., 2010)

OBJECTIVES

cloning of the rice genes of interest for rice

transformation

expression analysis of the rice genes under

Hirschmaniella oryzae infection by q-PCR

MATERIALS AND METHOD

Cloning

RNA extraction from 2 week old rice roots and cDNA

synthesized

amplification of concerned genes by PCR

purified PCR products cloned and sequenced

Sequences confirmed by

http://www.ncbi.nlm.nih.gov/VecScreen/VecScreen.h

tml

http://rice.plantbiology.msu.edu/index.shtml

MATERIALS AND METHODS…

Infection Experiment

80 well grown 16 days old rice seedlings

40 plants infected by nematode @ 205/plants

40 plants for control(not to be infected)

MATERIALS AND METHODS…

5 plants considered as 1 pool

32 total samples of root and shoot tissues collected

from infected & uninfected plants at 1, 3, 5 and 7 dpi

RNA extracted, cDNA synthesized

Quality check of cDNA sample and qPCR primers

q-PCR

data obtained were analysed by REST-2009 software

RESULTS AND DISCUSSION

CLONING

Amplification of DEAD, purine and Cupin

Amplification of rice genes with 52ºC

annealing temperature for 40 cycles Amplification of rice genes with 45ºC

annealing temperature for 5 cycles

followed by 52ºC for 35 cycles

Cupin was successfully cloned

Rice genes expression pattern

Relative expression level of three rice genes in root tissues at different

time points after H. oryzae infection (infected vs non-infected plants)

Cupin

Cupin highly up regulated at 3 dpi in root tissue under

H. oryzae infection (Kyndt, 2011. unpublished data)

Cupin up-regulated at 2 dpi in Nipponbare rice under

Striga hermonthica infection (Swarbrick et al.,2008) and also

under infection with Magnaporthe oryzae (Marcel et al.,

2010).

Cupin domain gene shows homology with a germin-

like protein from Barley (http://rice.plantbiology.msu.edu/cgi-

bin/ORF_infopage.cgi)

GLPs in basal host resistance against powdery mildew

(Blumeria graminis spp.) in barley and wheat (Christensen et al., 2004; Zimmermann et al, 2006).

GLP governing gene chr8 OsGLP contributes broad

resistance against two rice fungal pathogens which

are causal agent of rice blast and sheath blight

diseases (Manosalva et al., 2009).

Membrane

down-regulation of Membrane at different time points

supports the unpublished finding of Kyndt (2011)

Membrane down-regulated in Nipponbare under

Striga hermonthica infection (Swarbrick et al., 2008)

B-box

B-box was down-regulated under H. oryzae infection

in the research done by Kyndt (2011).

Mukhopadhyay et al. (2003) have reported that

transgenic tobacco over-expressing a zinc-finger

protein gene from rice conferred tolerance to cold,

dehydration, and salt stress.

CONCLUSION AND SUGGESTIONS

Cupin domain gene may have role in basal defense

against H. oryzae

Membrane protein and B-box zinc finger family protein

also may have significant role during rice and the

nematode interaction.

all three genes have systemic activation in whole plant

under H. oryzae infection

these genes can be over-expressed or silenced in rice

and their impact on the nematode population and its

infectivity can be observed.

THANK YOU

FOR YOUR

KIND ATTENTION

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http://rice.plantbiology.msu.edu/cgi-bin/ORF_infopage.cgi

LIST OF PRIMERS USED TO AMPLIFY GENES AND IN

COLONY PCR

Name Locus F-primer R-primer

DEAD LOC_Os11g44910 atggcggggtacgagagg tcaagagggaatctttatgcagttg

Purine LOC_Os03g08880 atggccaccattactgctgc ctaaggcgccgctgactc

Cupin LOC_Os08g09080 atggcttcgtcttccttcc tcagtaatggttgttctcccag

Membrane LOC_Os04g33830.1 atggccgcctccaccgtctc tcacttgtcgaagtagccctc

Ring LOC_Os04g50100 atggcatcctctgctcctgc tcacattggatgatctgaatc

B-box LOC_Os02g39360.1 atgaagatccagtgcgacgcgt tcaaccaagatcagggacga

SP6 taatacgactcactatagggcgaattgg

T7 atttaggtgacactatagaatactcaagc

OligodT tttttttttttttttttttttttttvn

LIST OF Q-PCR PRIMERS

Gene name Locus F-primer R-primer

Cupin LOC_Os08g09080 tgtgttcgtattccctgtgg agggttctggctgctaagtg

Member LOC_Os04g33830.1 gctcttcttctccagcatcg caggaacagcccaaggtg

Ring LOC_Os04g50100 gacgttctgcaggaatccat cccttcccttgctgttctc

B-box LOC_Os02g39360.1 cctccagttctccgactacg ttgtggaacaggtcgatgtc

EXP LOC_Os03g27010 tgtgagcagcttctcgtttg tgttgttgcctgtgagatcg

EXPnarsai LOC_Os11g21990.1 aggaacatggagaagaacaagg cagaggtggtgcagatgaaa

EIF5C LOC_Os07g02340.1 cacgttacggtgacacctttt gacgctctccttcttcctcag

Genes 1 dpi samples 3 dpi samples 5 dpi samples 7 dpi samples

Cupin Bl S1 Ho S2 Bl R1 Ho S2

Membrane Bl R2 Ho R1 Bl S2 Ho R2

Ring Ho R1 Bl S1 Ho S1 Bl R1

B-box Ho S2 Bl R2 Ho R2 Bl S2

LIST OF RANDOMLY SELECTED CDNA SAMPLES TO CHECK

Q-PCR PRIMERS

Bl: uninfected; Ho: infected; S: shoot; R: root; dpi: day/s post infection