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The blast resistance gene Pi54of cloned from Oryza officinalis interacts with Avr-Pi54 through its novel non-LRR domains.

Devanna NB, Vijayan J, Sharma TR - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, India.

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.

No MeSH data available.


In vitro expression and characterization of Pi54of protein.a: SDS-PAGE analysis of IPTG induced BL21 clones. Lane 2–7: Pi54of clones; Lane 8–13: Pi54 clones. b: Western blot of Pi54of (1–3) and Pi54 (4–6) protein using polyclonal Ab developed for Pi54 protein. c: Purified Pi54of protein. d: MS mass spectra MALDI-TOF analyzed Pi54of protein; ID 1233.555 showed match with Pi54 protein.
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pone-0104840-g005: In vitro expression and characterization of Pi54of protein.a: SDS-PAGE analysis of IPTG induced BL21 clones. Lane 2–7: Pi54of clones; Lane 8–13: Pi54 clones. b: Western blot of Pi54of (1–3) and Pi54 (4–6) protein using polyclonal Ab developed for Pi54 protein. c: Purified Pi54of protein. d: MS mass spectra MALDI-TOF analyzed Pi54of protein; ID 1233.555 showed match with Pi54 protein.

Mentions: Protein electrophoresis of IPTG induced and uninduced pET29a clones confirmed the induction of ∼43 kDa and ∼37 kDa protein products in Pi54of and Pi54 clones, respectively (Figure 5). Western blot analysis revealed the hybridization of antibody to ∼43 kDa Pi54of and ∼37 kDa Pi54 proteins (Figure 5). The peptide mass fingerprinting analysis performed using MALDI-TOF mass spectrometry showed match of its spectra with the Pi54 proteins of rice lines resistant to the pathogen (Figure 5).


The blast resistance gene Pi54of cloned from Oryza officinalis interacts with Avr-Pi54 through its novel non-LRR domains.

Devanna NB, Vijayan J, Sharma TR - PLoS ONE (2014)

In vitro expression and characterization of Pi54of protein.a: SDS-PAGE analysis of IPTG induced BL21 clones. Lane 2–7: Pi54of clones; Lane 8–13: Pi54 clones. b: Western blot of Pi54of (1–3) and Pi54 (4–6) protein using polyclonal Ab developed for Pi54 protein. c: Purified Pi54of protein. d: MS mass spectra MALDI-TOF analyzed Pi54of protein; ID 1233.555 showed match with Pi54 protein.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4128725&req=5

pone-0104840-g005: In vitro expression and characterization of Pi54of protein.a: SDS-PAGE analysis of IPTG induced BL21 clones. Lane 2–7: Pi54of clones; Lane 8–13: Pi54 clones. b: Western blot of Pi54of (1–3) and Pi54 (4–6) protein using polyclonal Ab developed for Pi54 protein. c: Purified Pi54of protein. d: MS mass spectra MALDI-TOF analyzed Pi54of protein; ID 1233.555 showed match with Pi54 protein.
Mentions: Protein electrophoresis of IPTG induced and uninduced pET29a clones confirmed the induction of ∼43 kDa and ∼37 kDa protein products in Pi54of and Pi54 clones, respectively (Figure 5). Western blot analysis revealed the hybridization of antibody to ∼43 kDa Pi54of and ∼37 kDa Pi54 proteins (Figure 5). The peptide mass fingerprinting analysis performed using MALDI-TOF mass spectrometry showed match of its spectra with the Pi54 proteins of rice lines resistant to the pathogen (Figure 5).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, India.

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.

No MeSH data available.