DUAL RNASeq ANALYSIS REVEALS AN INCREASE OF FUNGAL PATHOGENICITY FUNCTIONS AND A ROLE OF PLANT GLUTAMINE SYNTHETASE IN NITROGEN-INDUCED SUSCEPTIBILITY TO THE RICE BLAST FUNGUS
ABSTRACT: In order to understand the mechanisms of Nitrogen induced susceptibility (NIS) we’ve conducted a dual RNAseq experiment on rice infected tissues by Magnaporthe oryzae. At 0 day post inoculation and 2 days post inoculation tissues have been collected on mock inoculated and M. oryzae inoculated plants. Rice were conducted under two type of nitrogen fertilization: 0N all fertilization but nitrogen, 1N all fertilization and NH4NO3. The fertilization was applied one day before inoculation. RNAseq was conducted both on rice and fungal RNA. Overall design: shoot tissus mRNA 28-day old rice plant were generated by deep sequencing. Three samples per conditions, Four conditions: 0N Mock, 0N Inoc, 1N Mock, 1N Inoc. 0N = plant fertilized one day before inoculation without nitrogen. 1N = plant fertilized one day before inoculation with NH4NO3. Mock = Plant inoculated with control solution (gelatin 0,5%). Inoc = Plant inoculated with Magnaporthe oryzae isolate Guy11 with gelatin 0,5%.
Project description:In order to understand the mechanisms of Drought induced susceptibility (DIS) we’ve conducted a dual RNAseq experiment on rice infected tissues by Magnaporthe oryzae. At 4 days post inoculation tissues have been collected on mock inoculated and M. oryzae inoculated plants. Rice were conducted under two type of water regime: DIS Drought during three days before inoculation, NoDIS no drought before inoculation. RNAseq was conducted both on rice and fungal RNA. Overall design: shoot tissus mRNA 21-day old rice plant were generated by deep sequencing. Three samples per conditions, Four conditions: NoDIS Mock, NoDIS Inoc, DIS Mock, DIS Inoc. NoDIS = plant were conducted with a normal water regime. DIS = plant received no watering during three days before inoculation. Mock = Plant inoculated with control solution (gelatin 0,5%). Inoc = Plant inoculated with Magnaporthe oryzae isolate Fr13 with gelatin 0,5%.
Project description:Rice blast (caused by Magnaporthe oryzae) is one of the most destructive diseases of rice. While many blast resistance (R) genes have been identified and deployed in rice cultivars, little is known about the R gene-mediated defense mechanism. We used a rice transgenic line harboring the resistance gene Piz-t to investigate the R gene-mediated resistance response to infection.We conducted comparative proteome profiling of the Piz-t transgenic Nipponbare line (NPB-Piz-t) and wild-type Nipponbare (NPB) inoculated with M. oryzae at 24, 48, 72 h post-inoculation (hpi) using isobaric tags for relative and absolute quantification (iTRAQ) analysis. Comparative analysis of the response of NPB-Piz-t to the avirulent isolate KJ201 and the virulent isolate RB22 identified 114 differentially expressed proteins (DEPs) between KJ201-inoculated NPB-Piz-t (KJ201-Piz-t) and mock-treated NPB-Piz-t (Mock-Piz-t), and 118 DEPs between RB22-inoculated NPB-Piz-t (RB22-Piz-t) and Mock-Piz-t. Among the DEPs, 56 occurred commonly in comparisons KJ201-Piz-t/Mock-Piz-t and RB22-Piz-t/Mock-Piz-t. In a comparison of the responses of NPB and NPB-Piz-t to isolate KJ201, 93 DEPs between KJ201-Piz-t and KJ201-NPB were identified. DEPs in comparisons KJ201-Piz-t/Mock-Piz-t, RB22-Piz-t/Mock-Piz-t and KJ201-Piz-t/KJ201-NPB contained a number of proteins that may be involved in rice response to pathogens, including pathogenesis-related (PR) proteins, hormonal regulation-related proteins, defense and stress response-related proteins, receptor-like kinase, and cytochrome P450. Comparative analysis further identified 7 common DEPs between the comparisons KJ201-Piz-t/KJ201-NPB and KJ201-Piz-t/RB22-Piz-t, including alcohol dehydrogenase I, receptor-like protein kinase, endochitinase, similar to rubisco large subunit, NADP-dependent malic enzyme, and two hypothetical proteins.Our results provide a valuable resource for discovery of complex protein networks involved in the resistance response of rice to blast fungus.
Project description:The transcriptomic modulations leading to defense response in rice one hour after inoculation by Xanthomonas oryzae pv oryzae. Xoo and mock inoculated plant of cultivars IET8585 (bacterial leaf blight resistant) and IR-24 (bacterial leaf blight susceptible) were compared.
Project description:Rice blast disease caused by a fungus Magnaporthae oryzae is one of the most important biotic factors that severely damage the rice crop. Several molecular approaches are now being applied to tackle this issue in rice. It is of interest to study long non-coding RNA (lncRNA) in rice to control the disease. lncRNA, a non-coding transcript that does not encode protein, is known to play an important role in gene regulation of various biological processes. Here we describe a computational pipeline to identify lncRNA from a resistant rice line. The number of lncRNA found in resistant line was 1429, 1927 and 1981 in mock and M. oryzae (ZB13 and Zhong) inoculated samples, respectively. Functional classification of these lncRNA reveals a higher number of long intergenic non-coding RNA compared to antisense lncRNA in both mock and M. oryzae inoculated resistant rice lines. Many intergenic lncRNA candidates were identified from resistant rice line and their role to regulate the resistance mechanism in rice during M. oryzae invasion is implied.
Project description:Worldwide, rice blast (Pyricularia oryzae) causes more rice crop loss than other diseases. Acid rain has reduced crop yields globally for nearly a century. However, the effects of acid rain on rice-Pyricularia oryzae systems are still far from fully understood. In this study, we conducted a lab cultivation experiment of P. oryzae under a series of acidity conditions as well as a glasshouse cultivation experiment of rice that was inoculated with P. oryzae either before (P. + SAR) or after (SAR + P.) simulated acid rain (SAR) at pH 5.0, 4.0, 3.0 and 2.0. Our results showed that the growth and pathogenicity of P. oryzae was significantly inhibited with decreasing pH treatments in vitro culture. The SAR + P. treatment with a pH of 4.0 was associated with the highest inhibition of P. oryzae expansion. However, regardless of the inoculation time, higher-acidity rain treatments showed a decreased inhibition of P. oryzae via disease-resistance related enzymes and metabolites in rice leaves, thus increasing disease index. The combined effects of high acidity and fungal inoculation were more serious than that of either alone. This study provides novel insights into the effects of acid rain on the plant-pathogen interaction and may also serve as a guide for evaluating disease control and crop health in the context of acid rain.
Project description:The present study was conducted to evaluate the potential of rice rhizosphere associated antagonistic bacteria for growth promotion and disease suppression of bacterial leaf blight (BLB). A total of 811 rhizospheric bacteria were isolated and screened against 3 prevalent strains of BLB pathogen Xanthomonas oryzae pv. oryzae (Xoo) of which five antagonistic bacteria, i.e., Pseudomonas spp. E227, E233, Rh323, Serratia sp. Rh269 and Bacillus sp. Rh219 showed antagonistic potential (zone of inhibition 1-19 mm). Production of siderophores was found to be the common biocontrol determinant and all the strains solubilized inorganic phosphate (82-116 μg mL-1) and produced indole acetic acid (0.48-1.85 mg L-1) in vitro. All antagonistic bacteria were non-pathogenic to rice, and their co-inoculation significantly improved plant health in terms of reduced diseased leaf area (80%), improved shoot length (31%), root length (41%) and plant dry weight (60%) as compared to infected control plants. Furthermore, under pathogen pressure, bacterial inoculation resulted in increased activity of defense related enzymes including phenylalanine ammonia-lyase and polyphenol oxidase, along with 86% increase in peroxidase and 53% increase in catalase enzyme activities in plants inoculated with Pseudomonas sp. Rh323 as well as co-inoculated plants. Bacterial strains showed good colonization potential in the rice rhizosphere up to 21 days after seed inoculation. Application of bacterial consortia in the field resulted in an increase of 31% in grain yield and 10% in straw yield over non-inoculated plots. Although, yield increase was statistically non-significant but was accomplished with overall saving of 20% chemical fertilizers. The study showed that Pseudomonas sp. Rh323 can be used to develop dual-purpose inoculum which can serve not only to suppress BLB but also to promote plant growth in rice.
Project description:Rice blast disease is a major threat to rice production worldwide, but the mechanisms underlying rice resistance to the causal agent Magnaporthe oryzae remain elusive. In this whole-genome transcriptome study of rice early defense response to M. oryzae, we applied Affymetrix Rice Genome Genechip to compare the compatible and incompatible rice-M. oryzae interactions in 24 hours post-inoculation. Overall design: Leaf samples were harvested from three biological replicates of fungal- and mock-inoculated seedlings at 24 hours post-inoculation, from which RNA were extracted and analyzed with Genechip Rice Genome Array.
Project description:Rice blast disease is a major threat to rice production worldwide, but the mechanisms underlying rice resistance to the causal agent Magnaporthe oryzae remain elusive. In this whole-genome transcriptome study of rice early defense response to M. oryzae, we applied Affymetrix Rice Genome Genechip to compare the compatible and incompatible rice-M. oryzae interactions in 24 hours post-inoculation. Leaf samples were harvested from three biological replicates of fungal- and mock-inoculated seedlings at 24 hours post-inoculation, from which RNA were extracted and analyzed with Genechip Rice Genome Array.
Project description:Rice (Oryza sativa L.) is the only widely cultivated gramineous crops that cannot be infected by rust fungi. To decipher the molecular basis of rice nonhost resistance (NHR) to Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust, proteomic analysis was performed using the two-dimensional electrophoresis (2-DE) technique. The expressed proteins from rice leaves 24 and 48 h post inoculation with Pst and from mock-inoculated leaves were identified. Quantitative analysis revealed a total of 27 differentially expressed proteins in response to Pst inoculation. Most of these proteins fall into the category "response to stimulus" and are involved in basic resistance processes, such as glycerol-3-phosphate and hydrogen peroxide signaling. A homologue of wheat leaf rust resistance protein Lr10 was also identified, implicating multiple layers of plant defense are implicated in rice NHR to Pst. These results demonstrate an intrinsic relationship between host and nonhost resistance. Changes in abundance of these proteins, together with their putative functions reveal a comprehensive profile of rice NHR to Pst and provide new insights into plant immunity.
Project description:The hemibiotrophic fungus Magnaporthe oryzae produces specialized biotrophic invasive hyphae (IH) that alter membrane structure and defense responses in invaded rice cells. IH successively invade live neighbor cells, apparently through plasmodesmata. Understanding fungal and rice genes that contribute to biotrophic invasion has been a challenge because so few plant cells have encountered IH at the earliest infection stages. Using a rice sheath inoculation method, we successfully enriched for infected tissue RNA that contained ~20% fungal RNA at a point when most IH were still growing in first-invaded rice cells. The RNAs were analyzed using the whole-genome M. oryzae oligoarray and a rice oligoarray. Rice genes that were induced >50-fold during infection were enriched for genes involved in transferring information from sensors to cellular responses. Fungal genes that were induced >50-fold in IH included the PWL2 avirulence gene and genes encoding hypothetical secreted proteins. The IH-specific secreted proteins are candidate effectors, proteins that the fungus secretes into live host cells to control cellular processes. Gene knock-out analyses of three putative effector genes failed to show major effects on pathogenicity. Details of the blast interaction transcriptome will provide insights on the mechanisms of biotrophic plant disease. Keywords: Disease state analysis Overall design: Our goal was to compare expression in infected rice sheath to expression in mock inoculated rice, and to compare expression in biotrophic IH to expression in mycelium grown in nutrient medium. Using the rice microarray (Agilent Technologies), we analyzed samples from three biological replicates of rice leaf sheath at 36 hours after inoculation with the rice blast fungus M. oryzae. The same samples were used with the Agilent M. oryzae microarray (see series GSE8517). To estimate the ratio of fungal to rice RNAs in the infected sheaths, we compared RT-PCR amplification of fungal genes in infected tissue to amplification in standards produced by mixing known ratios of pure mycelial RNA with mock-inoculated rice RNA. Using this assay, fungal RNA content in infected tissues were approximately 20% of the total RNAs. We prepared control mixtures by pooling 20% mycelial RNA and 80% RNA from mock-inoculated rice sheath. Complementary RNAs from infected tissues were labeled with Cy3 or Cy5 and hybridized together with the control mixture RNA labeled with the other dye. Three independent biological replications were performed, with separate hybridizations for 2 technical replications and corresponding dye swap experiments. Data were analyzed by Rosetta Resolver® and showed a correlation of over 80% between biological replications.