Characterizing functional role of G-proteins in nonhost

disease resistance in rice (Oryza sativa)

Seonghee Lee1, Hee-Kyung Lee1, Hiroshi Hisano2, Yasuhiro Ishiga1, Zengyu Wang2 and Kirankumar S. Mysore1

Abstract

Rice (Oryza sativa) is one of the most important food crops in the world and a model system of monocotyledon plants for many biological researches. The complete genome sequence of rice is available for functional genomics study. In our previous study, we found that several G-proteins are involved in nonhost disease resistance against bacterial pathogens in Arabidopsis, tomato, and N. benthamiana. To further investigate the mechanisms of G-proteins in rice, we silenced two G-protein homologues (G-ch6 and G-ch7) in rice using RNA interference (RNAi). A japonica rice cultivar, Nipponbare, whose genome has been sequenced, was used for Agrobacterium-mediated transformation. We are characterizing how downregulation of the G-protein gene expression affect nonhost disease resistance in rice.

1 Plant Biology Division, 2 Forage Improvement Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, Oklahoma 73401 USA.

Nonhost resistance of rice to cereal rust

Nonhost disease resistance (NHR) is one of the most important

plant defense mechanisms against pathogen attack. It has

been well known that rice is immune to fungal rust pathogens,

but the mechanism is still largely unknown.

Recently, we identified and functionally characterized several

important G-proteins that are involved in nonhost disease

resistance in Arabidopsis. To investigate the functional role of

these G-proteins in rice nonhost resistance, we silenced two G-

protein homologues (G-ch6 and G-ch7) in rice using RNAi

(RNA interference). The purposes of the present study were to

examine the level of down-regulation of rice G-proteins in

transgenic rice plants and determine how silencing these genes

in rice affect nonhost resistance against rust pathogens.

Quick guide for Agrobacterium-mediated

transformation of rice

1. Nishimura, A., Aichi, I. and Matsuoka, M. 2006. A protocol for Agrobacterium-mediated transformation in rice. Nature Protocols 1(6): 2796-2802.

2. Ayliffe, M., Devilla, R., Mago, R., White, R., Talbot, M., Pryor, A. and Leung, H. 2011. Nonhost resistance of rice to rust pathogens. 24:1143–1155.

Agrobacterium-mediated transformation is one of most widely

used methods for genetic modification in rice. Recently, an

efficient method for Agrobacterium-mediated transformation

was developed in rice. This new method is able to reduce the

regeneration time and increase transformation efficiency. Using

this procedure with minor modifications, we obtained transgenic

rice plants in 2 months with high transformation efficiency.

RNAi-mediated gene silencing in rice

Agrobacterium-mediated transformation with RNAi vector

GUS/attR2 (Site A) GUS/attR2 (Site D)

After transforming pANDA-G-protein into A. tumefaciens

EHA105, the agrobacteria carrying the RNAi vector construct

were confirmed by PCR using a primer amplifying GUS region

with another gene specific primer. Also, regenerated transgenic

rice plants carrying the RNAi construct were confirmed using

this primer set and primers for hygromycin gene.

Seed sterilization

Rice nonhost resistance to rusts is NOT a consequence of basic incompatibility. It involves induced defenses and restricting growth of rust pathogens (Ayliffe et al., 2011)

RICE WHEAT

Role of the G-protein involved in nonhost

disease resistance in Arabidopsis

0

0.4

0.8

1.2

G-ch6 G-ch7

Re

lative

ge

ne

exp

ressio

n

Nipponbare G-RNAi

Seed incubation Embryogenic calli

generation Transformation

1st Selection 2nd Selection

© 2010 by The Samuel Roberts Noble Foundation, Inc.

Shoot regeneration Rooting and growth

G-proteins are involved in stomata-based defense and

apoplast defense in Arabidopsis

Response of switchgrass rust pathogen

(Puccinia emaculata) inoculation in rice

To determine the downregulation of two G-proteins (G-ch6 and

G-ch7), RNA was extracted from RNAi lines and wild-type (cv.

Nipponbare). qRT-PCR was performed using an internal control

of OsUBQ5. The levels of downregulation were 50% to 80% for

G-ch6 and 40% to 60 % for G-ch7. The growth of transgenic

plant was inhibited when both G-proteins are greatly down-

regulated (especially for G-ch7) compared with wild-type

(Nipponbare).

Reference

wild-type

g-protein

Pstab (nonhost) Psm (host)

KCl-MES

wild-type

g-protein

ABA flg22 Pstab

Epidermal peels of wild-type (Col-0) and g-protein mutant

leaves were treated with stomata opening buffer (KCl-MES),

ABA, flg22, and a nonhost pathogen, Pseudomonas syringae

pv. tabaci. g-protein mutation impairs the regulation of stomatal

closure in response to ABA, PAMPs, and Pstab.

Plants were inoculated with

P. syringae pv. maculicola

(Psm; host) and Pstab

(nonhost) by syringe

infiltration in Arabidopsis. Disease symptoms were developed in g-protein mutant after

nonhost bacterial inoculation. wild-type

(Nipponbare)

G-RNAi

Infection of rice leaf tissue by P. emaculata. Leaf tissues were

collected 4 days after inoculation and WTG-stained for imaging.

Germination of a P. emaculata urediniospores (Sp) produced a

germ tube (Gt) in WT and G-RNAi, but appressoria (Ap) were

only found on the surface of a G-RNAi leaves. Stomata were

present near the appressorium.