Cloning and functional validation of early inducible Magnaporthe oryzae responsive CYP76M7 promoter from rice

Cloning and functional characterization of plant pathogen inducible promoters is of great significance for their use in the effective management of plant diseases. The rice gene CYP76M7 was up regulated at 24, 48, and 72 hours post inoculation (hpi) with two isolates of Magnaporthe oryzae Mo-ei-11 and Mo-ni-25. In this study, the promoter of CYP76M7 gene was cloned from rice cultivar HR-12, characterized and functionally validated. The Transcription Start Site of CYP76M7 was mapped at 45 bases upstream of the initiation codon. To functionally validate the promoter, 5′ deletion analysis of the promoter sequences was performed and the deletion fragments fused with the β-glucuronidase (GUS) reporter gene were used for generating stable transgenic Arabidopsis plants as well as for transient expression in rice. The spatial and temporal expression pattern of GUS in transgenic Arabidopsis plants and also in transiently expressed rice leaves revealed that the promoter of CYP76M7 gene was induced by M. oryzae. The induction of CYP76M7 promoter was observed at 24 hpi with M. oryzae. We report that, sequences spanning -222 bp to -520 bp, with the cluster of three W-boxes, two ASF1 motifs and a single GT-1 element may contribute to the M. oryzae inducible nature of CYP76M7 promoter. The promoter characterized in this study would be an ideal candidate for the overexpression of defense genes in rice for developing durable blast resistance rice lines

Marker-assisted enhancement of bacterial blight (Xanthomonas oryzae pv. oryzae) resistance in a salt-tolerant rice variety for sustaining rice production of tropical islands

IntroductionBacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae is a major disease of rice, specially in the tropical regions of the world. Developing rice varieties with host resistance against the disease is the most effective and economical solution for managing the disease.MethodsPyramiding resistance genes (Xa4, xa5, xa13,and Xa21) in popular rice varieties using marker-assisted backcross breeding (MABB) has been demonstrated as a cost-effective and sustainable approach for establishing durable BB resistance. Here, we report our successful efforts in introgressing four resistance genes (Xa4, xa5, xa13, and Xa21) from IRBB60 to CARI Dhan 5, a popular salt-tolerant variety developed from a somaclonal variant of Pokkali rice, through functional MABB.Results and discussionBoth BB and coastal salinity are among the major challenges for rice production in tropical island and coastal ecosystems. Plants with four, three, and two gene pyramids were generated, which displayed high levels of resistance to the BB pathogen at the BC3F2 stage. Under controlled salinity microplot environments, the line 131-2-175-1223 identified with the presence of three gene pyramid (Xa21+xa13+xa5) displayed notable resistance across locations and years as well as exhibited a salinity tolerance comparable to the recurrent parent, CARI Dhan 5. Among two BB gene combinations (Xa21+xa13), two lines, 17-1-69-334 and 46-3-95-659, demonstrated resistance across locations and years, as well as salt tolerance and grain production comparable to CARI Dhan 5. Besides salinity tolerance, five lines, 17-1-69-179, 46-3-95-655, 131-2-190-1197, 131-2-175-1209, and 131-2-175-1239, exhibited complete resistance to BB disease. Following multilocation testing, potential lines have been identified that can serve as a prospective candidate for producing varieties for the tropical Andaman and Nicobar Islands and other coastal locations, which are prone to BB and coastal salinity stresses

Artificial Seed Production of Tylophora indica for Interim Storing and Swapping of Germplasm

Our research work demonstrates the single bead alginate-encapsulation, interim storing and conversion of Tylophora indica (Burm. Fil.) Merrill. Most effective encapsulation of in vitro nodal segments [(4 ± 1) mm long], ensuing in sphere-shaped artificial seeds of similar morphology, was achieved through 75 mmol⋅L−1 calcium chloride (CaCl2 ⋅ 2H2O) plus 3% (w/v) Na-alginate with 93.3% conversion frequency. The earliest conversion (within 7 days of incubation) of artificial seeds occurred in half-strength liquid Murashige and Skoog medium. Among the three different temperature regimes [(5 ± 1) °C, (15 ± 1) °C, and (25 ± 1) °C], storage of artificial seeds at (15 ± 1) °C executed the highest frequency of conversion (90%) after 15 days of storage. Lengthier storage significantly reduced the conversion frequency of artificial seeds irrespective of storage temperature. Nevertheless, the conversion frequency after 30 days of storage at (15 ± 1) °C was recorded at 70% without further decline even following 45 days of storage, which evidently suggests that lower temperature (15 ± 1) °C is apt for storage and subsequent conversion of T. indica artificial seeds. The present protocol could be expedient for short-term storing and swapping of T. indica germplasms between national and international laboratories

Possible molecular mechanism of <i>Pi54of</i> gene.

<p>Avr-Pi54 binds to Pi54of protein (blue) at the non-LRR region upstream to the LRR domain. Pi54of may perceive pathogen signals through STI1 which acts as an anchor for the so called defensome complex involving Os Rac1 (Rac/Rop GTPase), RACK1A (Receptor of Activated C Kinase), RAR (Required for Mla12 Resistance), SGT1 (Suppressor of the G2 allele of skp1), MAPK6 (a rice Mitogen-Activated Protein Kinase), Rboh (NADPH oxidases).</p

<i>In vitro</i> expression and characterization of Pi54of protein.

<p>a: SDS-PAGE analysis of IPTG induced BL21 clones. Lane 2–7: <i>Pi54of</i> clones; Lane 8–13: <i>Pi54</i> clones. b: Western blot of <i>Pi54of</i> (1–3) and <i>Pi54</i> (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.</p

Phylogeny and alignment of amino acid residues from ORF of <i>Pi54, Pi54of</i> and <i>Pi54rh</i> orthologues.

<p>a: Phylogenetic relationship among the cloned rice blast resistance genes. Tree was generated using neighbor-joining method by MEGA.6 software with bootstrap value 1000 replications. The unit of branch length is 0.1 nucleotide substitutions per site, and is indicated by a bar at the bottom left corner of the tree. b: Aligned sequences of Pi54 protein orthologues using BioEdit tool.</p

Molecular docking of Pi54 proteins.

<p>Predicted tertiary structures of Pi54 orthologue protein Pi54 (a); Pi54rh (b); Pi54of (c); Pi54tp (d) and AVR-Pi54 protein (e) generated by <i>ab initio</i> modeling using I-TASSER server. Various secondary structure; Alpha helices (blue), β-sheets (red), Coils (green) have been depicted. Docked AVR-Pi54 with protein Pi54 (f); Pi54rh (g); Pi54of (h) and Pi54tp (i) using Z-DOCK server in Discovery studio 3.5. In the interaction, AVR protein is depicted in pink color and R-protein in blue color.</p

Docking features (hydrogen bond forming residues with bond length <4Å) of modelled Pi54 rthologues and AVR-Pi54.

<p>Docking features (hydrogen bond forming residues with bond length <4Å) of modelled Pi54 rthologues and AVR-Pi54.</p

Comparison of various energy values and docking features for each of the best of modelled complex of Pi54 orthologues and AVR-Pi54 using ZDOCKER in DS3.5.

<p>Binding energy = energy of complex − [energy (receptor) + energy (ligand)].</p