Abstract
The dominant rice blast resistance gene Pi54 cloned by map-based cloning approach from indica rice cultivar Tetep confers broad spectrum resistance to Magnaporthe oryzae. In this investigation, an orthologue of Pi54 designated as Pi54of was cloned from Oryza officinalis conferring high degree of resistance to M. oryzae and is functionally validated. We have also characterized the Pi54of protein and demonstrate its interaction with AVR-Pi54 protein. The Pi54of encoded ∼43 kDa small and unique cytoplasmic LRR family of disease resistance protein having unique Zinc finger domain overlapped with the leucine rich repeat regions. Pi54of showed Magnaporthe-induced expression. The phylogenetic and western blot analysis confirmed orthologous nature of Pi54 and Pi54of genes, whereas the identity of protein was confirmed through MALDI-TOF analysis. The in silico analysis showed that Pi54of is structurally more stable than other cloned Pi54 proteins. The molecular docking revealed that Pi54of protein interacts with AVR-Pi54 through novel non-LRR domains such as STI1 and RhoGEF. The STI1 and GEF domains which interact with AVR-Pi54 are also components of rice defensome complex. The Pi54of protein showed differential domain specificity while interacting with the AVR protein. Functional complementation revealed that Pi54of transferred in two rice lines belonging to indica and japonica background imparts enhanced resistance against three highly virulent strains of M. oryzae. In this study, for the first time, we demonstrated that a rice blast resistance gene Pi54of cloned from wild species of rice provides high degree of resistance to M. oryzae and might display different molecular mechanism involved in AVRPi54-Pi54of interaction.
Highlights
Rice is the staple food for larger part of the world population
Expression analysis and cloning of Pi54of gene The phenotyping results showed that the wild rice O. officinalis was completely resistant when inoculated with M. oryzae isolate Mo ei-11, whereas the susceptible check rice line HR12 showed scale 4–5 susceptible reaction (Figure 1)
The 59 RACE PCR results revealed the position of Transcription Start Site (TSS) at 76-bp upstream of the start codon (ATG) of the Pi54of transcript (Figure S1)
Summary
Rice is the staple food for larger part of the world population. The major biotic stress of rice which is considered as one of the most damaging diseases worldwide is rice blast caused by hemibiotrophic fungi Magnaporthe oryzae. The adaptive nature of M. oryzae is mainly because of breakdown in host resistance due to mutations in the pathogen leading to instability of avirulence (AVR) genes [4]. The fungus can overcome even major R gene-mediated resistance with the mutation in the corresponding AVR gene limiting the life span of major R-genes [5,6]. This necessitates time bound release of resistant varieties for effective management of rice blast. In view of this, continued identification and cloning of new effective genes and their alleles are critical for R-gene mediated management of rice blast disease
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