Os11g0639100

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Annotated Information

This gene(Pikh; Pi54; Pi54rh) confers a high degree of resistance to diverse strains of the fungus Magnaporthe oryzae. The rice blast resistance gene Pi-kh has been isolated from the indica rice line Tetep showing resistance to different M. oryzae strains in the North-Western Himalayan region of India .

Function

Pi54 (Pi-kh cloned from the rice line Tetep) confers broad spectrum resistance against geographically diverse strains of M. oryzae collected from various parts of India and the US . In order to develop sustainably successful blast-resistant rice lines, a comprehensive dissection of the reactions downstream of R–Avr interactions is highly desirable.The co-expression patterns of bacterial disease resistance genes and their transcriptional regulators in transgenic rice have indicated that the resistance genes trigger an immune response . The expression patterns of defence response genes involved in rice–M. oryzae interaction have been widely studied .

The transgenic lines continually show a high degree of resistance and hypersensitive response to the blast pathogen in the fifth generation as well. Typical blast lesions as observed in the non-transgenic susceptible rice line Taipei 309 (TP). No such disease symptoms were observed in the resistant transgenic line (TP-Pi54); hypersensitive response (HR) was observed in the TP-Pi54 line.

Adopting a stringent criterion for the expression fold change value (FCA) of >2.0 and the P value <0.05, a total of 1154 differentially expressed genes were identified in TP-Pi54 plants. Of these, 587 were up-regulated, whereas 567 genes were found to be down-regulated. All the genes that showed differential expression in the transgenic blast-resistant line TP-Pi54 were clustered and functionally annotated. The level of expression of these genes in TP-Pi54 plants was compared with that in TP plants. Based on K-means clustering, the functionally annotated genes were classified into eight different clusters. These genes were also analysed on the basis of their Gene Ontology (GO) score into three main GO categories, including biological process (BP), molecular function (MF), and cellular component (CC).

The number of genes was finalized using the filtering criteria of fold change >2.0 and P-value correction <¼0.05 by FDR (Benjamini-Hochberg). Genes were classified into 16 different categories based on Gene Ontology. The number of up- and down-regulated genes for each functional category is shown in the histogram. The functional categories are: A, binding; B, catalytic activity; C, transporter activity; D, transcription regulator activity; E, molecular transducer activity; F, antioxidant activity; G, metabolic processes; H, cellular processes; I, biological regulation; J, establishment of localization; K, response to stimulus; L, anatomical structure function; M, cell part; N, organelle; O, organelle part; and P, molecular complex. (A) Up-regulated genes, (B) Down-regulated genes.

Expression

All the putative transformants (T0) were first screened for the presence of transgene by PCR. Genomic DNA was isolated from the leaves of putative transformants by using DNeasy Plant Mini Kit (QIAGEN, Cologne, USA) as per manufacturer’s instructions. Three primer sets were designed for the screening of transformants by PCR (Fig. 1a). The first pair of primer, CaPi-F: GAGGAGGTTTCCCGATATTAC and CaPi-R: GGTAGGTTCTCCAACCATTCTG was se- lected to get amplification of the region between CaMV35S promoter and Pi54 gene with an amplicon size of 1.43 kb. The second pair HyPi-F: CGGTGAGTTCAGGCTTTTTC and Hypi-R: TGCAGTGCTCTCAATTTTGG was designed from within hpt gene to give an amplification of 1 kb. The third pair GUS-F: ATGGTAGATCTGAGGGG and GUS-R: AAGTCGAAGTTCGGCT was designed from within the β-glucuronidase gene with an amplicon of 750 bp.

Evolution

Phylogenetic analysis of the Pi54rh with 19 blast resistance genes cloned from rice. Bootstrap values, corresponding to the match times of branching orders (1,000 replicates), are shown at the nodes of each branch point. The unit of branch length is 0.2 nucleotide substitutions per site, as indicated by a bar at the bottom left corner of the tree.

Structural components of Pi54 orthologues. a Nipponbare; b Tetep ; c O. rhizomatis and d tertiary structure of PI54RH protein generated by homology modelling using Modeller module of Accelrys Discovery Studio Software. CC domain (sky blue), NBS domain (parrot green), LRR domains (violet) and Zn-finger domain (yellow), N indicates amino terminal and C indicates carboxy terminal of PI54RH protein.

Detection of callose deposition

To analyze this important aspect of defense response by the rice plants and understand about the possible involvement of Pi54 gene in the deposition of callose, 15 m thick transverse sections of leaf epidermis were prepared from the transgenic and non-transgenic plants after 0, 72, 96 and 120 hpi and stained with callose specific aniline blue stain. Stained sections were observed under fluorescent light microscope. Careful histochemical examination of these sections revealed the gradually increasing deposition of callose resulting into thickened cell walls in the plants of transgenic line TP-Pi54-2, which has earlier been found to be highly resistant to M. oryzae.

Labs working on this gene

1. National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute,India.

1. Department of Biotechnology, Himachal Pradesh University, India.