1.4.12
THE RANK1 GENE THAT DIFFERENTIALLY EXPRESSES IN BLAST-RESISTANT RICE PLANT ENCODES A NOVEL PROTEIN CONTAINING ANKYRIN REPEATS

CHAOZU HE, S FONG and GUO-LIANG WANG

Institute of Molecular Agrobiology, Singapore

Background and objectives
When a plant is attacked by a pathogen, it can in most cases fend off the infection by mounting a battery of defence responses. The activation of plant defence occurs upon pathogen recognition and results in the halt of pathogen ingress. Systemic acquired resistance (SAR) is one of the important components of this complex system that plants use to defend themselves from pathogens. SAR can be triggered by a local HR to an avirulent pathogen, which renders uninfected parts of the plant resistant to a variety of normally virulent pathogens. SAR is a particularly important aspect of plant-pathogen response because it is a pathogen-inducible, systematic resistance against a broad spectrum of pathogens. Significant progress has been made recently in deciphering molecular aspects of SAR. The Arabidopsis gene NPR1/NIM1 has been cloned using a map-based cloning strategy [1, 2]. The gene encodes a novel protein containing ankyrin repeats implicated in protein-protein interaction.

Rice blast, an often devastating disease caused by Magnaporthe grisea, occurs in most rice-growing areas worldwide. The disease reduces rice yield significantly, particularly in the temperate flooded and tropical upland rice ecosystems. Many major genes for resistance have been identified and widely used in breeding programmes. However, the molecular mechanism of the host defence to this pathogen is mostly unknown. The objective of this research is to isolate gene(s) involving the defence pathway in rice against blast infection.

Results and discussion
A Pi-2 near-isogenic line C101A51 and the concurrent parent CO39 were used in this experiment. Using the differential display strategy, a cDNA fragment (about 600 bp) was isolated which was observed only in the inoculated resistant plants. Northern blot analysis indicated that this gene was strongly induced as early as 4 h post-inoculation. Sequence analysis revealed that this cDNA fragment has homology to those proteins containing ankyrin repeats including the Arabidopsis gene NPR1/NIM1 and mammalian gene family I-kB. This partial cDNA clone was used as the probe to screen a BAC library made from an Indica cultivar IR64. Six positive BAC clones were identified and mini-prepped for further subcloning. A 5-kb genomic clone which contains RANK1 has been sequenced. A 1.22-kb full-length cDNA has been obtained by RT-PCR. It encodes a 340 aa protein and contains seven exons. Southern blot analysis revealed that restriction fragment length polymorphism exists between C101A51 and CO39 as well as other cultivars.

Ankyrin repeat-containing proteins have been shown to have diverse functions and to be involved in protein-protein interactions. Some of these proteins in mammals are transcription-regulating proteins, such as the NF-kB and its inhibitor I-kB. In Arabidopsis, NPR1/NIM1 which is homologous to the NF-kB inhibitor I-kB controls the onset of systemic acquired resistance. The transcription factor targeted by NPR1/NIM1 serves as a repressor of SAR gene expression and disease resistance either by direct and indirect action [2]. Since the RANK1 gene is similar to the Arabidopsis gene NPR1/NIM1, we speculate that it may be involved in the rice SAR defence mechanism to blast infection. However, more experiments are needed to prove this hypothesis. Firstly, Northern blot analysis of RANK1's expression in cultivars carrying different resistance genes post-inoculation and in SA/BTH-treated plants will be conducted. Secondly, transgenic plants transformed with an antisense construct or a cDNA construct driven by the 35S promoter will be generated to further investigate the function of RANK1 in rice.

Reference
1. Cao H, Glazebrook J, Clarke JD et al., 1997. Cell 88, 57-63.
2. Ryals J, Weymann K, Lawton K et al., 1997. Plant Cell 9, 425-439.