Blast Inoculation Research Articles

Genome-Wide Association Study Identifies a Rice Panicle Blast Resistance Gene Pb3 Encoding NLR Protein.

Rice blast is a worldwide fungal disease that seriously affects the yield and quality of rice. Identification of resistance genes against rice blast disease is one of the effective ways to control this disease. However, panicle blast resistance genes, which are useful in the fields, have rarely been studied due to the difficulty in phenotypic identification and the environmental influences. Here, panicle blast resistance-3 (Pb3) was identified by a genome-wide association study (GWAS) based on the panicle blast resistance phenotypes of 230 Rice Diversity Panel I (RDP-I) accessions with 700,000 single-nucleotide polymorphism (SNP) markers. A total of 16 panicle blast resistance loci (PBRLs) within three years including one repeated locus PBRL3 located in chromosome 11 were identified. In addition, 7 genes in PBRL3 were identified as candidate genes by haplotype analysis, which showed significant differences between resistant and susceptible varieties. Among them, one nucleotide-binding domain and Leucine-rich Repeat (NLR) gene Pb3 was highly conserved in multiple resistant rice cultivars, and its expression was significantly induced after rice blast inoculation. Evolutionary analysis showed that Pb3 was a typical disease resistance gene containing coiled-coil, NB-ARC, and LRR domains. T-DNA insertion mutants and CRISPR lines of Pb3 showed significantly reduced panicle blast resistance. These results indicate that Pb3 is a panicle blast resistance gene and GWAS is a rapid method for identifying panicle blast resistance in rice.

OsVTC1-1 Gene Silencing Promotes a Defense Response in Rice and Enhances Resistance to Magnaporthe oryzae

Rice blast disease is a serious disease in rice caused by Magnaporthe oryzae (M. oryzae). Ascorbic acid (AsA), or vitamin C, is a strong antioxidant that prevents oxidative damage to cellular components and plays an essential role in plant defense response. GDP-D-mannose pyrophosphorylase (GMP or VTC1) is an enzyme that generates GDP-D-mannose for AsA, cell wall, and glycoprotein synthesis. The OsVTC1 gene has three homologs in the rice genome: OsVTC1-1, OsVTC1-3, and OsVTC1-8. Using OsVTC1-1 RNAi lines, this study investigated the role of the OsVTC1-1 gene during rice blast fungus inoculation. The OsVTC1-1 RNAi inoculated with rice blast fungus induced changes to cell wall monosaccharides, photosynthetic efficiency, reactive oxygen species (ROS) accumulation, and malondialdehyde (MDA) content. Additionally, the OsVTC1-1 RNAi lines were shown to be more resistant to rice blast fungus than the wild type. Genes and proteins related to defense response, plant hormone synthesis, and signaling pathways, especially salicylic acid and jasmonic acid, were up-regulated in the OsVTC1-1 RNAi lines after rice blast inoculation. These results suggest that the OsVTC1-1 gene regulates rice blast resistance through several defense mechanisms, including hormone synthesis and signaling pathways.

Interactive effects of tropospheric ozone and blast disease (Magnaporthe oryzae) on different rice genotypes

Rising tropospheric ozone concentrations can cause rice yield losses and necessitate the breeding of ozone-tolerant rice varieties. However, ozone tolerance should not compromise the resistance to important biotic stresses such as the rice blast disease. Therefore, we investigated the interactive effects of ozone and rice blast disease on nine different rice varieties in an experiment testing an ozone treatment, blast inoculation, and their interaction. Plants were exposed to an ozone concentration of 100 ppb for 7 h per day or ambient air throughout the growth period. Half of the plants were simultaneously infected with rice blast inoculum. Grain yield was significantly reduced in the blast treatment (17%) and ozone treatment (37%), while the combination of both stresses did not further decrease grain yields compared to ozone alone. Similar trends occurred for physiological traits such as vegetation indices, normalized difference vegetation index (NDVI), photochemical reflectance index (PRI), Lichtenthaler index 2 (Lic2), and anthocyanin reflectance index 1 (ARI1), as well as stomatal conductance and lipid peroxidation. Ozone exposure mitigated the formation of visible blast symptoms, while blast inoculation did not significantly affect visible ozone symptoms. Although different genotypes showed contrasting responses to the two types of stresses, no systematic pattern was observed regarding synergies or trade-offs under the two types of stresses. Therefore, we conclude that despite the similarities in physiological stress responses to ozone and blast, the tolerance to these stresses does not appear to be genetically linked in rice.

The OsOXO2, OsOXO3 and OsOXO4 Positively Regulate Panicle Blast Resistance in Rice

BackgroundAlthough panicle blast is more destructive to yield loss than leaf blast in rice, the cloned genes that function in panicle blast resistance are still very limited and the molecular mechanisms underlying panicle blast resistance remain largely unknown.ResultsIn the present study, we have confirmed that the three Oxalate oxidase (OXO) genes, OsOXO2, OsOXO3 and OsOXO4 from a blast-resistant cultivar BC10 function in panicle blast resistance in rice. The expression of OsOXO2, OsOXO3 and OsOXO4 were induced by panicle blast inoculation. Subcellular localization analysis revealed that the three OXO proteins are all localized in the nucleus and cytoplasm. Simultaneous silencing of OsOXO2, OsOXO3 and OsOXO4 decreased rice resistance to panicle blast, whereas the OsOXO2, OsOXO3 and OsOXO4 overexpression rice plants individually showed enhanced panicle blast resistance. More H2O2 and higher expression levels of PR genes were observed in the overexpressing plants than in the control plants, while the silencing plants exhibited less H2O2 and lower expression levels of PR genes compared to the control plants. Moreover, phytohormone treatment and the phytohormone signaling related gene expression analysis showed that panicle blast resistance mediated by the three OXO genes was associated with the activation of JA and ABA signaling pathways but suppression of SA signaling pathway.ConclusionOsOXO2, OsOXO3 and OsOXO4 positively regulate panicle blast resistance in rice. The OXO genes could modulate the accumulation of H2O2 and expression levels of PR gene in plants. Moreover, the OXO genes mediated panicle blast resistance could be regulated by ABA, SA and JA, and may be associated with the activation of JA and ABA signaling pathways but suppression of the SA signaling pathway.

NAC transcription factor ONAC066 positively regulates disease resistance by suppressing the ABA signaling pathway in rice

This is the first time to dissect the mechanism of NACs-mediated disease resistance in plants using metabolomic approach and discover the involvement of ABA signaling pathway in NACs-mediated disease resistance. NAC transcription factors have been validated as important regulators in stress responses, but their molecular mechanisms in plant disease resistance are still largely unknown. Here we report that the NAC gene ONAC066 (LOC_Os01g09550) is significantly activated by rice blast infection. ONAC066 is ubiquitously expressed and this protein is localized in the nucleus. Overexpression of ONAC066 quantitatively enhances resistance to blast disease and bacterial blight in rice. The transcript levels of PR genes are also dramatically induced in ONAC066 overexpressing plants. Exogenous abscisic acid (ABA) strongly activates the transcription of ONAC066 in rice. Further analysis shows that overexpression of ONAC066 remarkably suppresses the expression of ABA-related genes, whereas there are no obvious differences for salicylic acid (SA) and jasmonic acid (JA)-related genes between wild-type and ONAC066 overexpressing plants. Consistently, lower endogenous ABA levels are identified in ONAC066 overexpressing plants compared with wild-type plants before and after blast inoculation, while no significant differences are observed for the SA and JA levels. Yeast one-hybrid assays demonstrate that ONAC066 directly binds to the promoters of LIP9 and NCED4 to modulate their expression. Moreover, the metabolomic study reveals that the ONAC066 overexpressing plants accumulated higher contents of soluble sugars and amino acids both before and after pathogen attack, when compared to wild-type plants. Taken together, our results suggest that ONAC066 positively regulates rice resistance to blast and bacterial blight, and ONAC066 exerts its functions on disease resistance by modulating of ABA signaling pathway, sugars and amino acids accumulation in rice.

Panicle blast 1 (Pb1) resistance is dependent on at least four QTLs in the rice genome

BackgroundRice blast is the most serious disease afflicting rice and there is an urgent need for the use of disease resistance (R) genes in blast tolerance breeding programs. Pb1 is classified as a quantitative resistance gene and it does not have fungal specificity. Pb1-mediated resistance develops in the latter stages of growth. However, some cultivars, such as Kanto209 (K209), cultivar name Satojiman, despite possessing Pb1, do not exert resistance to rice blast during the reproductive stage.ResultsWe found that the expression of WRKY45 gene downstream of Pb1 was weakly induced by rice blast inoculation at the full heading stage in K209. Genetic analysis using the SNP-based Golden Gate assay of K209 crossing with Koshihikari Aichi SBL (KASBL) found at least four regions related to the resistance in the rice genome (Chr8, Chr9, Chr7, Chr11). Mapping of QTL related to Chr7 confirmed the existence of factors that were required for the resistance of Pb1 in the 22 to 23 Mbp region of the rice genome.ConclusionWe clarified how the K209 cultivar is vulnerable to the blast disease despite possessing Pb1 and found the DNA marker responsible for the quantitative resistance of Pb1. We identified the QTL loci required for Pb1-mediated resistance to rice panicle blast. Pb1 was negatively dependent on at least three QTLs, 7, 9 and 11, and positively dependent on one, QTL 8, in the K209 genome. This finding paves the way for creating a line to select optimal QTLs in order to make use of Pb1-mediated resistance more effectively.

Endoplasmic reticulum membrane-bound MoSec62 is involved in the suppression of rice immunity and is essential for the pathogenicity of Magnaporthe oryzae.

Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) constitutes the first line of plant inducible immunity. As an important step of plant colonization, phytopathogens have to suppress PTI, and secreted effectors are therefore co-evolved and deployed. In this study, we characterized the function of MoSec62 of Magnaporthe oryzae, the causal agent of the destructive rice blast. MoSec62 encodes a homologue of Sec62p, a yeast endoplasmic reticulum (ER) membrane transporter for precursors of secretory proteins. We showed that a T-DNA insertion into the promoter region of MoSec62, causing a disturbance to the up-regulation of MoSec62 expression during blast invasion, resulted in a complete loss of blast virulence of the mutant, M1575. Both 3,3'-diaminobenzidine (DAB) staining of the infected rice leaves and expression analysis revealed that the infectious attempt by the mutant led to strong defence responses of rice. Consistently, in transcriptomic analysis of rice leaves subject to blast inoculation, a battery of defence responses was found to be induced exclusively on M1575 challenge. For further exploration, we tested the pathogenicity on a highly susceptible rice variety and detected the accumulation of Slp1, a known PTI suppressor. Both results suggested that the mutant most likely failed to overcome rice PTI. In addition, we showed that MoSec62 was able to rescue the thermosensitivity of a yeast Δsec62, and the MoSec62-GFP fusion was co-localized to the ER membrane, both suggesting the conservation of Sec62 homologues. In conclusion, our data indicate that MoSec62, probably as an ER membrane transporter, plays an essential role in antagonizing rice defence at the early stages of blast invasion.

A Standardized Inoculation Protocol to Test Wheat Cultivars for Reaction to Head Blast Caused by Magnaporthe oryzae (Triticum pathotype)

Wheat blast, caused by the Triticum pathotype of Magnaporthe oryzae, poses a significant threat to wheat production worldwide. Because this pathotype does not occur in the United States, it is important to prepare for its possible introduction. As part of this preparation, over 500 U.S. wheat cultivars were tested for susceptibility or resistance to head blast. Inoculations were conducted under biosafety level 3 conditions. However, the protocols to phenotype wheat cultivars vary among researchers, and head blast inoculation protocols need to be standardized so that results can be easily interpreted and shared internationally. The protocol presented, based on several years' experience, is recommended for common use to facilitate direct comparison of disease phenotyping results among researchers. Accepted for publication 12 August 2016.

Rice blast resistance gene Pikahei-1(t), a member of a resistance gene cluster on chromosome 4, encodes a nucleotide-binding site and leucine-rich repeat protein

Pikahei-1(t) is the strongest quantitative trait locus (QTL) for blast resistance in upland rice cv. Kahei, which has strong field resistance to the rice blast disease. A high-quality bacterial artificial chromosome library was used to fine-map Pikahei-1(t) within ~300 kb on the 31-Mb region on rice chromosome 4. Of the 42 predicted open reading frames, seven resistance gene analogs (RGAs) with the nucleotide-binding site and leucine-rich repeat (NBS-LRR) domain were identified. Among these, RGA1, 2, 3, 5, and 7, but not RGA4 and 6, were found to be expressed in Kahei and monogenic lines containing Pikahei-1(t). Blast inoculation of transgenic rice lines carrying the genomic fragment of each RGA revealed that only RGA3 was associated with blast resistance. On the basis of these results, we concluded that RGA3 is the Pikahei-1(t) and named it Pi63. Pi63 encoded a typical coiled-coil-NBS-LRR protein and showed isolate-specificity. These results suggest that Pi63 behaves like a typical Resistance (R) gene, and the strong and broad-spectrum resistance of Kahei is dependent on natural pyramiding of multiple QTLs. The blast resistance levels of Pi63 were closely correlated with its gene expression levels, indicating a dose-dependent response of Pi63 function in rice resistance. Pi63 is the first cloned R gene in the R gene cluster on rice chromosome 4, and its cloning might facilitate genomic dissection of this cluster region.

Resistance spectrum assay and fine mapping of the blast resistance gene from a rice experimental line, IRBLta2-Re

In the present study, we performed the resistance assessment by rice blast inoculation on IRBLta2-Re and IRBLta-CP1, the experimental lines supposed to carry rice blast resistance genes Pita2 and Pita, respectively. The analysis by using 196 rice blast isolates derived from China indicated that the resistance spectrum of IRBLta2-Re was broader than that of IRBLta-CP1. Both IRBLta2-Re and IRBLta-CP1 have the Pita gene by analyzing the functional single amino acid difference of Pita/pita locus. To identify the additional gene in IRBLta2-Re, 1250 F2 individuals from the cross between CO39 and IRBLta2-Re were used as the mapping population. The F2 population was inoculated with the blast isolate 08-T4 which was incompatible to IRBLta2-Re, but compatible to CO39 and IRBLta-CP1. In the phenotypic data analysis, the F2 population segregated in a 3:1 ratio for resistant and susceptible plants, respectively, suggesting that IRBLta2-Re has an additional resistance gene other than Pita, which was tentatively designated Pita3(t) (supposed to be Pita2). To identify the Pita3(t), a total of 50 microsatellite and 12 position specific microsatellite markers distributed by two sides of the Pita gene were selected in the parent polymorphism screening. The results showed that PT4 and PT5 were co-segregated with the target gene. A contig map corresponding to the resistance gene and Pita genes was constructed based on the fine mapping and bioinformatics assay. The resistance gene, Pita3(t), was, thus, assumed to be in an interval of approximately 178 kb which containing a total of 5 NBS–LRR genes, and was about 500 kb away from the Pita gene.

Comparative Transcriptome Profiling of the Early Response to Magnaporthe oryzae in Durable Resistant vs Susceptible Rice (Oryza sativa L.) Genotypes

Durable resistance to blast, the most significant fungal disease of rice, represents an agronomically relevant character. Gigante Vercelli (GV) and Vialone Nano (VN) are two old temperate japonica Italian rice cultivars with contrasting response to blast infection: GV displays durable and broad resistance while VN is highly susceptible. RNA-seq was used to dissect the early molecular processes deployed during the resistance response of GV at 24 h after blast inoculation. Differential gene expression analysis identified 1,070 and 1,484 modulated genes, of which 726 and 699 were up regulated in response to infection in GV and VN, respectively. Gene ontology (GO) enrichment analyses revealed a set of GO terms enriched in both varieties but, despite this commonality, the gene sets contributing to common GO enriched terms were dissimilar. The expression patterns of genes grouped in GV-specific enriched GO terms were examined in detail including at the transcript isoform level. GV exhibited a dramatic up-regulation of genes encoding diterpene phytoalexin biosynthetic enzymes, flavin-containing monooxygenase, class I chitinase and glycosyl hydrolase 17. The sensitivity and high dynamic range of RNA-seq allowed the identification of genes critically involved in conferring GV resistance during the early steps of defence perception-signalling. These included chitin oligosaccharides sensing factors, wall associated kinases, MAPK cascades and WRKY transcription factors. Candidate genes with expression patterns consistent with a potential role as GV-specific functional resistance (R) gene(s) were also identified. This first application of RNA-seq to dissect durable blast resistance supports a crucial role of the prompt induction of a battery of responses including defence-related genes as well as members of gene families involved in signalling and pathogen-related gene expression regulation.

Stress repressive expression of rice SRZ1 and characterization of plant SRZ gene family

A zinc finger protein gene was cloned from rice and designated as SRZ1 ( stress repressive zinc finger protein 1). SRZ1 encodes a protein with three C2C2-type zinc finger motifs that are structurally similar to human ZNF265 zinc finger motifs. SRZ1 was expressed with high level in leaves and markedly repressed by salt, cold, drought and abcisic acid stresses but not by salicylic acid and blast inoculation in rice seedlings. Ectopic expression of SRZ1 in tobacco plants repressed expression of abiotic stress-related genes including osmotin, NtERB10B, NtERB10C, and increased plant sensitivity to cold and salt stresses. Database searches showed SRZ genes were widely distributed among plant species and their expressions in rice and Arabidopsis were significantly down-regulated by at least one type of abiotic stresses. This study indicates that SRZ1 and its relatives in plants may play negative roles in abiotic stress signaling and the down-regulation of them might be crucial for plant tolerance against stresses.

Expression of a Magnaporthe grisea Elicitor and Its Biological Function in Activating Resistance in Rice

The expression of a protein elicitor from Magnaporthe griesea and its biological function in activating resistance in rice ( Oryza sativa L) were reported. The gene of elicitor was expressed in Escherichia coli cells and produced a His 6 -fusion protein with 42 kD apparent molecular weight on SDS-PAGE. The purified protein could induce the resistance to blast disease, with the control efficiency of 46.47% and 36.41% at the 14 th day and the 21 st day after blast inoculation, respectively. After treatment with the expressed protein, the phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities were promoted in rice plants, meanwhile, the transcription levels of STKM, FAD, PBZ1 and PR1 genes were increased in rice plants. Moreover, after comparing the profile of total rice leaf proteins on two-dimensional electrophoresis gel, about 14 proteins were found to be increased in expression level after the expressed protein treatment. All the results indicated that the expressed protein could act as an elicitor to trigger the resistance in rice.

Transcriptomic analyses of space-induced rice mutants with enhanced susceptibility to rice blast

Mutagenic factors of the space environment influence organisms in different aspects. To elucidate the transcriptomic effects of space flight, a space flight-induced rice mutant, 972-4, and its on-ground control, 972ck, were inoculated with rice blast pathogens. Compared to the control, the mutant exhibited reduced resistance to the rice blast pathogen CH45. Microarray technique was employed to analyze affected genes and revealed that 481 genes were expressed at higher levels in the mutant strain and 188 genes were expressed at higher levels in the control strain under normal growth conditions, indicating that transcriptomic changes of rice seeds are induced by the space environment. After inoculation with the rice blast pathogen CH45, however, 2680 genes were differentially expressed in 972ck and 1863 genes were differentially expressed in 972-4. In addition, disease evaluation indicated that the control strain 972ck is more resistant to the rice blast pathogen CH45 than mutant strain 972-4. In addition, genes in both strains that were co-regulated after blast inoculation account for only 36.8% and 53.3% of the genes expressed in 972ck and 972-4, respectively. A large percentage of blast-regulated genes were not consistently expressed in 972-4 and 972ck, and the mutant and control strains exhibit different gene expression patterns after blast inoculation. Interestingly, 84 genes constitutively expressed higher in 972ck were up-regulated by blast inoculation, and 105 genes that were expressed at constitutively higher levels in 972-4 were down-regulated by blast inoculation. Of the differentially expressed, 7 encoded genes associated with pathogen resistance. Taken together, our results suggest that gene expression patterns are different between a space flight-induced rice mutant and its on-ground control, and the differential expression of resistance genes may be a potential mechanism that modulates the resistance of 972-4 to rice blast. Our results also suggest that the rice plants are suitable plant models for further research of the effects of the space environment on gene expression and function.

Simultaneous Expression of the Protease Inhibitors in a Rice Blast-Resistant Mutant

We have previously identified genes for four different protease inhibitors (PIs) that were induced upon rice blast infection in a rice blast resistant mutant SHM-11. Our expression analysis of the PIs indicated that induction of the PIs was the highest 24 hr after rice blast inoculation in the rice mutant SHM-11. Three PIs in the group of serine PIs were highly expressed while a cystein PI was weakly expressed upon rice blast inoculation. Four PIs were weakly induced 48 hr after pathogen inoculation in rice blast susceptible wild type rice plant. The simultaneous expression of three serine PIs was apparent from SHM-11 and two of them were induced in rice blast resistant Taebaegbyeo. One of them was induced in rice blast resistant Hwayeongbyeo while none of them were expressed in rice blast susceptible Nagdongbyeo and rice blast resistant Dongjinbyeo. Our results suggest that the expression of PI gene is rice cultivar specific and may be linked with the rice blast resistance in a specific rice mutant by the simultaneous expression of the PI genes.

BWMK1, a novel MAP kinase induced by fungal infection and mechanical wounding in rice.

The activation of the mitogen-activated protein (MAP) kinases by different environmental stresses has been previously observed in several dicot plant species. Here, we report the isolation of a novel MAP kinase in rice that is induced during infection by the blast fungus Magnaporthe grisea or upon mechanical wounding. The gene is designated as BWMK1 for blast- and wound-induced MAP kinase. The cDNA of BWMK1 was isolated from rice leaves challenged by the blast pathogen. Transcripts of the corresponding gene accumulated in rice leaves 4 h after blast inoculation and 30 min after mechanical wounding. This gene encodes a 506 amino acid protein that contains a new dual-phosphorylation activation motif TDY and about 150 unique amino acids on its C terminus. In-gel kinase activity and immunoprecipitation assays confirmed that BWMK1 is a functional MAP kinase. These results show that BWMK1 is a new member of the plant MAP kinase family and may mediate both defense and wound signaling in rice.