Grisea Infection Research Articles

Genome-wide characterization of the NBLRR gene family provides evolutionary and functional insights into blast resistance in pearl millet (Cenchrusamericanus (L.) Morrone).

The investigation is the first report on genome-wide identification and characterization of NBLRR genes in pearl millet. We have shown the role of gene loss and purifying selection in the divergence of NBLRRs in Poaceae lineage and candidate CaNBLRR genes for resistance to Magnaporthe grisea infection. Plants have evolved multiple integral mechanisms to counteract the pathogens' infection, among which plant immunity through NBLRR (nucleotide-binding site, leucine-rich repeat) genes is at the forefront. The genome-wide mining in pearl millet (Cenchrus americanus (L.) Morrone) revealed 146 CaNBLRRs. The variation in the branch length of NBLRRs showed the dynamic nature of NBLRRs in response to evolving pathogen races. The orthology of NBLRRs showed a predominance of many-to-one orthologs, indicating the divergence of NBLRRs in the pearl millet lineage mainly through gene loss events followed by gene gain through single-copy duplications. Further, the purifying selection (Ka/Ks < 1) shaped the expansion of NBLRRs within the lineage of pear millet and other members of Poaceae. Presence of cis-acting elements, viz. TCA element, G-box, MYB, SARE, ABRE and conserved motifs annotated with P-loop, kinase 2, RNBS-A, RNBS-D, GLPL, MHD, Rx-CC and LRR suggests their putative role in disease resistance and stress regulation. The qRT-PCR analysis in pearl millet lines showing contrasting responses to Magnaporthe grisea infection identified CaNBLRR20, CaNBLRR33, CaNBLRR46 CaNBLRR51, CaNBLRR78 and CaNBLRR146 as putative candidates. Molecular docking showed the involvement of three and two amino acid residues of LRR domains forming hydrogen bonds (histidine, arginine and threonine) and salt bridges (arginine and lysine) with effectors. Whereas 14 and 20 amino acid residues of CaNBLRR78 and CaNBLRR20 showed hydrophobic interactions with 11 and 9 amino acid residues of effectors, Mg.00g064570.m01 and Mg.00g006570.m01, respectively. The present investigation gives a comprehensive overview of CaNBLRRs and paves the foundation for their utility in pearl millet resistance breeding through understanding of host-pathogen interactions.

Genome-wide identification and characterization of NBLRR genes in finger millet (Eleusine coracana L.) and their expression in response to Magnaporthe grisea infection

BackgroundThe nucleotide binding site leucine rich repeat (NBLRR) genes significantly regulate defences against phytopathogens in plants. The genome-wide identification and analysis of NBLRR genes have been performed in several species. However, the detailed evolution, structure, expression of NBLRRs and functional response to Magnaporthe grisea are unknown in finger millet (Eleusine coracana (L.) Gaertn.).ResultsThe genome-wide scanning of the finger millet genome resulted in 116 NBLRR (EcNBLRRs1-116) encompassing 64 CC-NB-LRR, 47 NB-LRR and 5 CCR-NB-LRR types. The evolutionary studies among the NBLRRs of five Gramineae species, viz., purple false brome (Brachypodium distachyon (L.) P.Beauv.), finger millet (E. coracana), rice (Oryza sativa L.), sorghum (Sorghum bicolor L. (Moench)) and foxtail millet (Setaria italica (L.) P.Beauv.) showed the evolution of NBLRRs in the ancestral lineage of the target species and subsequent divergence through gene-loss events. The purifying selection (Ka/Ks < 1) shaped the expansions of NBLRRs paralogs in finger millet and orthologs among the target Gramineae species. The promoter sequence analysis showed various stress- and phytohormone-responsive cis-acting elements besides growth and development, indicating their potential role in disease defence and regulatory mechanisms. The expression analysis of 22 EcNBLRRs in the genotypes showing contrasting responses to Magnaporthe grisea infection revealed four and five EcNBLRRs in early and late infection stages, respectively. The six of these nine candidate EcNBLRRs proteins, viz., EcNBLRR21, EcNBLRR26, EcNBLRR30, EcNBLRR45, EcNBLRR55 and EcNBLRR76 showed CC, NB and LRR domains, whereas the EcNBLRR23, EcNBLRR32 and EcNBLRR83 showed NB and LRR somains.ConclusionThe identification and expression analysis of EcNBLRRs showed the role of EcNBLRR genes in assigning blast resistance in finger millet. These results pave the foundation for in-depth and targeted functional analysis of EcNBLRRs through genome editing and transgenic approaches.

Proteomic Analysis Reveals Salicylic Acid as a Pivotal Signal Molecule in Rice Response to Blast Disease Infection.

Rice blast disease caused by a fungus, Magnaporthe grisea, is one of the most destructive diseases in rice production worldwide, and salicylic acid (SA) can efficiently decrease the damage of M. grisea. Here, we combined the 2-Dimensional-Liquid Chromatography and the Matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (2D-LC-MALDI-TOF-TOF MS) techniques to compare and identify differentially expressed labelled proteins by the isobaric tags for relative and absolute quantitation (iTRAQ) between the blast-resistant cultivar Minghui and the susceptible rice cultivar Nipponbare in response to blast fungus infection. The group samples were treated with salicylic acid and compared to control samples. A total of 139 DEPs from the two cultivars showed either more than a two-fold change or alternating regulation patterns. Protein functionality analysis also exhibited that these proteins are involved in a wide range of molecular functions including: energy-related activity (30%), signal transduction (11%), redox homeostasis (15%), amino acid and nitrogen metabolism (4%), carbohydrate metabolism (5%), protein folding and assembly (10%), protein hydrolysis (9%), protein synthesis (12%), and other unknown functions (4%). Specifically, we demonstrated that exogenous treatment with salicylic acid promoted recovery in both rice cultivars from Magnaporthe grisea infection by enhancing: the regulation of signal transduction, increasing energy conversion and production through the regulation of the glycolytic pathway, and other various biochemical processes. These findings may facilitate future studies of the molecular mechanisms of rice blast resistance.

Metabolic Mechanism of Plant Defense against Rice Blast Induced by Probenazole.

The probenazole fungicide is used for controlling rice blast (Magnaporthe grisea) primarily by inducing disease resistance of the plant. To investigate the mechanism of induced plant defense, rice seedlings were treated with probenazole at 15 days post emergence, and non-treated plants were used for the control. The plants were infected with M. grisea 5 days after chemical treatment and incubated in a greenhouse. After 7 days, rice seedlings were sampled. The metabolome of rice seedlings was chemically extracted and analyzed using gas chromatography and mass spectrum (GC-MS). The GC-MS data were processed using analysis of variance (ANOVA), principal component analysis (PCA) and metabolic pathway elucidation. Results showed that probenazole application significantly affected the metabolic profile of rice seedlings, and the effect was proportionally leveraged with the increase of probenazole concentration. Probenazole resulted in a change of 54 metabolites. Salicylic acid, γ-aminobutyrate, shikimate and several other primary metabolites related to plant resistance were significantly up-regulated and some metabolites such as phenylalanine, valine and proline were down-regulated in probenazole-treated seedlings. These results revealed a metabolic pathway of rice seedlings induced by probenazole treatment regarding the resistance to M. grisea infection.

OsWRKY93 Dually Functions Between Leaf Senescence and in Response to Biotic Stress in Rice.

Cross talking between natural senescence and cell death in response to pathogen attack is an interesting topic; however, its action mechanism is kept open. In this study, 33 OsWRKY genes were obtained by screening with leaf aging procedure through RNA-seq dataset, and 11 of them were confirmed a significant altered expression level in the flag leaves during aging by using the reverse transcript quantitative PCR (RT-qPCR). Among them, the OsWRKY2, OsWRKY14, OsWRKY26, OsWRKY69, and OsWRKY93 members exhibited short-term alteration in transcriptional levels in response to Magnaporthe grisea infection. The CRISPR/Cas9-edited mutants of five genes were developed and confirmed, and a significant sensitivity to M. oryzae infection was observed in CRISPR OsWRKY93-edited lines; on the other hand, a significant resistance to M. oryzae infection was shown in the enhanced expression OsWRKY93 plants compared to mock plants; however, enhanced expression of other four genes have no significant affection. Interestingly, ROS accumulation was also increased in OsWRKY93 enhanced plants after flg22 treatment, compared with the controls, suggesting that OsWRKY93 is involved in PAMP-triggered immune response in rice. It indicated that OsWRKY93 was involved in both flag leaf senescence and in response to fungi attack.

Synchronised regulation of disease resistance in primed finger millet plants against the blast disease

Plants, being sessile, are exposed to an array of abiotic and biotic stresses. To adapt towards the changing environments, plants have evolved mechanisms that help in perceiving stress signals wherein phytohormones play a critical role. They have the ability to network enabling them to mediate defense responses. These endogenous signals, functioning at low doses are a part of all the developmental stages of the plant. Phytohormones possess specific functions as they interact with each other positively or negatively through cross-talks. In the present study, variations in the amount of phytohormones produced during biotic stress caused due to Magnoporthe grisea infection was studied through targeted metabolomics in both primed and control finger millet plants. Histochemical studies revealed callose deposition at the site of pathogen entry in the primed plants indicating its role during plant defense. The knowledge on the genetic makeup during infection was obtained by quantification of MAP kinase kinases 1 and 2 (MKK1/2) and lipoxygenase (LOX) genes, wherein the expression levels were high in the primed plants at 6 hours post-inoculation (hpi) compared to mock-control. Studies indicate the pivotal role of mitogen-activated protein kinase (MAPK or MAP kinases) during defense signalling. It is the first report to be studied on MAPK role in finger millet-blast disease response. Temporal accumulation of LOX enzyme along with its activity was also investigated due to its significant role during jasmonate synthesis in the plant cells. Results indicated its highest activity at 12 hpi. This is the first report on the variation in phytohormone levels in fingermillet - M. grisea pathosystem upon priming which were substantiated through salicylic acid (SA) pathway.

Evaluation of finger millet genotypes for partial resistance to leaf blast caused by Pyricularia grisea

Blast of finger millet (ragi) caused by Pyricularia grisea is disastrous disease to limit the potential yield levels in the farmers field. The pathogen affects the crop at all growth stages and the most susceptible stage for leaf blast is seedling stage. Use of resistant cultivars for minimizing losses due to leaf blast at early stage of crop growth is a viable and economical alternative. In the present study, partial resistance to leaf blast in fourteen genotypes of finger millet was assessed under field conditions. None of the genotypes were highly resistant to leaf blast. However, two genotypes namely PR 10-30 and VL 368 were shown resistance. Seven genotypes namely VR 988, VL 353, VL 348, TNAU 1214, GPU 45, VL 369, KOPM 942 were found moderately resistant, three namely PPR 2773, KRI 007-01, VR 708 were susceptible and two namely TNAU 1226 and REC 69 were highly susceptible. The correlation coefficients between leaf blast severities (LBS) with different components of partial resistance of slow blasting were significant. Low value of coefficient of infection (CI), apparent infection rate (r), area under disease progress curve (AUDPC), lesion number (LN), lesion area (LA), lesion circumference (LC) and high value of incubation period (IP) were recorded in resistant genotypes of finger millet, whereas, higher values of CI, r, AUDPC, LN, LA, LC and low values of IP were recorded in highly susceptible genotypes. A significant positive correlation was found in leaf blast severity with CI (r=0.948*), r (r=0.829*), AUDPC (r=0.998*), LN (r=0.967*), LA (r=0.957*), LC (r=0.976*) whereas IP (r=-0.825*) was negatively correlated with blast resistance. PR 10-30 and VL 368 showed higher degree of partial resistance or slow blasting resistance against P. grisea infection on finger millet.

Characterization and mapping of a novel light-dependent lesion mimic mutant lmm6 in rice (Oryza sativa L.)

A novel rice lesion mimic mutant (LMM) was isolated from an ethane methyl sulfonate (EMS)-induced 02428 mutant bank. The mutant, tentatively designated as lmm6, develops necrotic lesions in the whole growth period along with changes in several important agronomic traits. We found that the initiation of the lesions was induced by light and cell death occurred in lmm6 accompanied with accumulation of reactive oxygen species (ROS). The lower chlorophyll content, soluble protein content and superoxide dismutase (SOD) activity, the higher malondialdehyde (MDA) content were detected in lmm6 than in the wild type (WT). Moreover, the observation by transmission electronic microscope (TEM) demonstrated that some organelles were damaged and the stroma lamella of chloroplast was irregular and loose in mesophyll cell of lmm6. In addition, lmm6 was more resistant than WT to rice blast fungus Magnaporthe grisea infection, which was consistent with increased expression of four genes involved in the defense-related reaction. Genetic analysis showed that mutant trait of lmm6 is inherited as a monogenic recessive nuclear gene located on the long arm of chromosome 6. Using simple sequence repeat (SSR) markers, the target gene was finally delimited to an interval of 80.8 kb between markers MM2359 and MM2370, containing 7 annotated genes. Taken together, our results provide the information to identify a new gene involved in rice lesion mimic, which will be helpful in clarifying the mechanism of cell death and disease resistance in rice.

From Biological Warfare to the Brighter Side of Rice Research

Rice (Orzya sativa L.) is an important staple food crop in Asia providing nourishment to half of the world’s population. With the global human population set to rise to over nine billion by 2050 (according to United Nations predictions, see http://esa. un.org/wpp/Other-Information/Press_Release_WPP2010.pdf ), there are increasing pressures to secure the production and productivity of this crop for future generations. In addition, because rice is also considered a model organism for monocotyledonous plants due to its relatively small and sequenced genome, considerable efforts are underway to better understand the biology of this crop plant. To celebrate some of these excellent advances Plant and Cell Physiology is proud to present a special collection of articles featuring the latest research on rice, covering topics such as disease resistance and trait biology, to the latest technologies being developed to further facilitate in-depth research into this agronomically important species. Due to its growth in moist, temperate climates, rice is susceptible to pathogen attack. With incidences of rice blast fungus (Magnaporthe grisea) infection significantly decreasing annual global yields, research into rice-pathogen interactions is becoming central to stabilising the productivity of this crop food. Innate immunity in rice can be triggered by two main pathways either involving recognition of pathogen-associated molecular patterns (PAMPS) such as chitin and sphingolipids, or highly variable microbial effector molecules (Chen and Ronald 2011). The small plasma membrane bound Rac/Rop GTPase, OsRac1 is pivotal for both PAMP-triggered immunity and effector-triggered immunity, which have been shown to lead to a range of responses including cell death, ROS production, activation of pathogen-related gene expression and phytoalexin biosynthesis (Chen et al. 2010, Kawasaki et al. 1999). On pages 740–754, Kim et al. investigate the downstream transcriptional signalling cascade involved in triggering the OsRac1 mediated immune response in rice. The authors identify a novel bHLH transcription factor, Rac Immunity 1 (RAI1), that acts as a key downstream component of OsRac1 signalling following M. grisea attack to regulate expression of the elicitor-responsive genes PAL1 and OsWRKY19. Further, RAI1 interacts in vivo with OsMAPK3/6 in the OsRac1 complex, thereby expanding the battalion of plant signalling components responding to the relentless attack by microbial and pathogenic microorganisms. In an effort to sustain rice as a reliable food crop, trait research studies designed to ameliorate agronomically important traits such as grain size, vivipary, uniform germination, and seedling vigour and establishment have become a hot focus in recent years (Martinez-Andujar et al. 2012, Nambara and Nonogaki, 2012). Rice grains are complex structures that are formed following double fertilisation. Intriguingly, as vital carriers of genetic information, they are able to remain dormant for required periods of time before germinating under favourable conditions to ensure plant survival to the next generation. Therefore, grains ought to seemingly possess a mechanism that enables them to perform such a task and successfully develop into an established seedling. In this issue, Sano et al. (see pages 687–698) applied transcriptional and translational inhibitors to rice seeds, then performed a proteomic analysis of these chemically modified materials to determine the stability and extent to which seed proteins can be synthesised from long-stored mRNAs. Their findings indicate that while mature grains contained levels of stored mRNAs sufficient to initiate germination events, de novo translation was essential for successful completion of rice seed germination. Furthermore, this study provides novel insight into how plants have the ability to select discrete long-lived mRNAs to be translated during early germination when others are targeted for degradation. Flowering is a key agronomic trait in plants with wider significant biological implications, as it represents the onset of the mature reproductive phase in the plant life-cycle. Like animals, plants also respond to seasonal cues, such as exposure to day length, and adjust their time to flowering accordingly such that double fertilisation and seed production occurs when stressful conditions are least likely to be encountered. In contrast to Arabidopsis thaliana, flowering time in rice is promoted by exposure to short days. In addition, rice photoperiodicity is also mediated by two regulators of flowering under long days: Grain number, plant height and heading date locus 7 (Ghd7) and Early heading date 1 (Ehd1), for which there are no clear orthologues present in Arabidopsis (Itoh et al. 2010). Despite these apparent differences, the Flowering Time (FT) orthologues appears to play a central role in flowering regulation (Simpson and Dean 2002) in both species. Another key player in controlling flowering time is Early flowering 3 (ELF3), which in Arabidopsis is required for maintaining circadian rhythms (Dixon et al. 2011). In this issue, two papers report work on an ELF3 homologue in rice, Ef7/Heading date 17 (HD17) (Matsubara et al. 2008, Yuan et al. 2009). The Editor-in-chief’s choice article by Saito et al. on pages 717–728, presents work on the characterisation of the ef7 mutant, which exhibits delayed flowering. Genetic

Identification of a cluster of PR4-like genes involved in stress responses in rice

PR4 proteins constitute a pathogenesis-related (PR) protein family with a conserved BARWIN domain. In this study, we analyzed PR4-homologous genes in rice ( Oryza sativa L.) and identified five putative PR4 genes designated as OsPR4a− e. The five PR4 genes are located in tandem on chromosome 11 and constitute a gene cluster with high sequence similarity to each other. The OsPR4 proteins have high sequence similarity to reported PR4 proteins from monocotyledonous species and are predicted to be class II PR4 proteins. Distinct diversification of plant PR4 proteins exists between monocotyledonous and dicotyledonous plants. Except for OsPR4e, which was not detected with any transcript, the other four OsPR4 genes showed diverse temporal–spatial expression patterns, and their expressions are responsive to Magnaporthe grisea infection. Interestingly, the OsPR4 genes are also responsive to abiotic stresses. Their expression levels were strongly induced by at least one of the stress treatments including drought, salt, cold, wounding, heat shock, and ultraviolet. The transcript levels of OsPR4 genes were also induced by some phytohormones such as abscisic acid and jasmonic acid. Transgenic rice with overexpression of OsPR4a showed enhanced tolerance to drought at both seedling and reproductive stages. We conclude that rice PR4 genes are also involved in abiotic stress responses and tolerance in addition to their responsiveness to pathogen attacks.

Induced Pib Expression and Resistance to Magnaporthe grisea are Compromised by Cytosine Demethylation at Critical Promoter Regions in Rice

Pib is a well-characterized rice blast-resistance gene belonging to the nucleotide binding site (NBS) and leucine-rich repeat (LRR) superfamily. Expression of Pib was low under non-challenged conditions, but strongly induced by the blast-causing fungal pathogen Magnaporthe grisea, thereby conferring resistance to the pathogen. It is generally established that cytosine methylation of the promoter-region often plays a repressive role in modulating expression of the gene in question. We report here that two critical regions of the Pib promoter were heavily CG cytosine-methylated in both cultivars studied. Surprisingly, induced expression of Pib by M. grisea infection did not entail its promoter demethylation, and partial demethylation by 5-azacytidine-treatment actually reduced Pib expression relative to wild-type plants. Accordingly, the blast disease-resistance was compromised in the 5'-azaC-treated plants relative to wild-type. In contrast, the disease susceptibility was not affected by the 5'-azaC treatment in another two rice cultivars that did not contain the Pib gene, ruling out effects of other R genes and non-specific genotoxic effects by the drug-treatment as a cause for the compromised Pib-conditioned blast-resistance. Taken together, our results suggest that promoter DNA methylation plays a novel enhancing role in conditioning high-level of induced expression of the Pib gene in times of M. grisea infection, and its conferred resistance to the pathogen.

Phytochromes Regulate SA and JA Signaling Pathways in Rice and Are Required for Developmentally Controlled Resistance to Magnaporthe grisea

Old leaves of wild-type rice plants (Oryza sativa L. cv. Nipponbare) are more resistant to blast fungus (Magnaporthe grisea) than new leaves. In contrast, both old and new leaves of the rice phytochrome triple mutant (phyAphyBphyC) are susceptible to blast fungus. We demonstrate that pathogenesis-related class 1 (PR1) proteins are rapidly and strongly induced during M. grisea infection and following exogenous jasmonate (JA) or salicylic acid (SA) exposure in the old leaves, but not in the new leaves of the wild-type. In contrast, the accumulation of PR1 proteins was significantly attenuated in old and new leaves of the phyAphyBphyC mutant. These results suggest that phytochromes are required for the induction of PR1 proteins in rice. Basal transcription levels of PR1a and PR1b were substantially higher in the wild-type as compared to the phyAphyBphyC mutant, suggesting that phytochromes also are required for basal expression of PR1 genes. Moreover, the transcript levels of genes known to function in SA- or JA-dependent defense pathways were regulated by leaf age and functional phytochromes. Taken together, our findings demonstrate that phytochromes are required in rice for age-related resistance to M. grisea and may indirectly increase PR1 gene expression by regulating SA- and JA-dependent defense pathways.

Effect of a Photo-synthetic Inhibitor on Tryptamine Pathway-mediated Sekiguchi Lesion Formation in Lesion Mimic Mutant of Rice Infected withMagnaporthe grisea

A lesion-mimic mutant of rice (cv. Sekiguchi-asahi) showed enhanced resistance to Magnaporthe grisea infection, thereby inducing Sekiguchi lesion (sl) formation and tryptamine accumulation under light. Both Sekiguchi lesion formation and tryptamine accumulation in leaves infected with M. grisea were inhibited by pretreatment with the photosynthetic inhibitor, 3-(3, 4-Dichlorophenyl)- 1,1 -dimethylurea (DCMU), which suppressed the gene expression of tryptophan decarboxylase (TDC), monoamine oxidase activity, H 2 O 2 generation and DNA fragmentation. Catalase activity was inhibited by M. grisea infection under light, but magnitude of the inhibition was reduced in leaves pretreated with DCMU. Furthermore, tryptophan accumulated in M. grisea-infected leaves under light but not in DCMU-pretreated ones. Interestingly, such DCMU inhibition was reduced in the presence of tryptophan. Our studies suggest that chloroplasts function as the inhibitor of anti-oxidant system such as catalase activity and the supplier of a precursor of tryptamine and tryptophan in the sl mutant infected with M. grisea.

Effect of glyphosate on tryptamine production and Sekiguchi lesion formation in rice infected with Magnaporthe grisea

When exposed to light, the Sekiguchi lesion (sl) rice mutant has an enhanced resistance to Magnaporthe grisea infection responsible for Sekiguchi lesion formation and tryptamine accumulation. Glyphosate [N-(phosphonomethyl) glycine] pretreatment suppressed Sekiguchi lesion formation and tryptamine accumulation in the sl mutant after M. grisea infection even under light. This inhibition by glyphosate was blocked by the supply of exogenous tryptophan, but not by exogenous phenylalanine. In glyphosate-pretreated leaves, 5-enol-pyruvyl-shikimate-3-phosphate synthase gene expression and tryptophan biosynthesis were significantly suppressed. During tryptophan starvation, catalase activity was maintained at a high level even under light, leading to the suppression of H2O2 generation and DNA fragmentation. These results show a strong relationship between the tryptophan and tryptamine pathways in the induction of light-enhanced resistance to M. grisea infection in the sl mutant.

Effect of Photosynthetic Inhibitors on Induction of Tryptamine Pathway‐mediated Resistance in Lesion Mimic Mutant of Rice Infected with Magnaporthe grisea

AbstractRice cv. Sekiguchi‐asahi was identified as a lesion mimic mutant from cv. Asahi. This mutant showed light‐enhanced resistance to Magnaporthe grisea infection, inducing Sekiguchi lesion (sl) formation and tryptamine accumulation. To elucidate the role of chloroplasts on the activation of tryptamine pathway, the effect of some photosynthetic inhibitors on light‐dependant Sekiguchi lesion formation, tryptamine accumulation, and enzyme activities was investigated. When detached leaf blades which had been pretreated with Linuron, 1, 10‐Phenanthroline or Poly‐l‐lysine were inoculated with M. grisea, respectively, Sekiguchi lesion formation and typtamine accumulation were suppressed even under light, suppressing gene expression of tryptophan decarboxylase and monoamine oxidase activity. On the other hand, catalase activity was significantly inhibited in control leaves infected with M. grisea under light, but kept at high level in chemical‐pretreated leaves. These results suggested that photosynthetic activity in chloroplasts was involved in inhibition of antioxidant sysytem and activation of tryptamine pathway responsible for light‐enhanced resistance in sl mutant to M. grisea infection.

Disease Resistance against Magnaporthe grisea is Enhanced in Transgenic Rice with Suppression of -3 Fatty Acid Desaturases

Linolenic acid (18:3) is the most abundant fatty acid in plant membrane lipids and is a source for various oxidized metabolites, called oxylipins. 18:3 and oxylipins play important roles in the induction of defense responses to pathogen infection and wound stress in Arabidopsis. However, in rice, endogenous roles for 18:3 and oxylipins in disease resistance have not been confirmed. We generated 18:3-deficient transgenic rice plants (F78Ri) with co-suppression of two omega-3 fatty acid desaturases, OsFAD7 and OsFAD8. that synthesize 18:3. The F78Ri plants showed enhanced resistance to the phytopathogenic fungus Magnaporthe grisea. A typical 18:3-derived oxylipin, jasmonic acid (JA), acts as a signaling molecule in defense responses to fungal infection in Arabidopsis. However, in F78Ri plants, the expression of JA-responsive pathogenesis-related genes, PBZ1 and PR1b, was induced after inoculation with M. grisea, although the JA-mediated wound response was suppressed. Furthermore, the application of JA methyl ester had no significant effect on the enhanced resistance in F78Ri plants. Taken together, our results indicate that, although suppression of fatty acid desaturases involves the concerted action of varied oxylipins via diverse metabolic pathways, 18:3 or 18:3-derived oxylipins, except for JA, may contribute to signaling on defense responses of rice to M. grisea infection.

Magnaporthe grisea infection triggers RNA variation and antisense transcript expression in rice.

Rice blast disease, caused by the fungal pathogen Magnaporthe grisea, is an excellent model system to study plant-fungal interactions and host defense responses. In this study, comprehensive analysis of the rice (Oryza sativa) transcriptome after M. grisea infection was conducted using robust-long serial analysis of gene expression. A total of 83,382 distinct 21-bp robust-long serial analysis of gene expression tags were identified from 627,262 individual tags isolated from the resistant (R), susceptible (S), and control (C) libraries. Sequence analysis revealed that the tags in the R and S libraries had a significant reduced matching rate to the rice genomic and expressed sequences in comparison to the C library. The high level of one-nucleotide mismatches of the R and S library tags was due to nucleotide conversions. The A-to-G and U-to-C nucleotide conversions were the most predominant types, which were induced in the M. grisea-infected plants. Reverse transcription-polymerase chain reaction analysis showed that expression of the adenine deaminase and cytidine deaminase genes was highly induced after inoculation. In addition, many antisense transcripts were induced in infected plants and expression of four antisense transcripts was confirmed by strand-specific reverse transcription-polymerase chain reaction. These results demonstrate that there is a series of dynamic and complex transcript modifications and changes in the rice transcriptome at the M. grisea early infection stages.

Expression Analysis of Rice Defense-Related Genes in Turfgrass in Response to Magnaporthe grisea

ABSTRACT Magnaporthe grisea (anamorph = Pyricularia grisea) causes blast on rice (Oryza sativa) and gray leaf spot on turfgrass. Gray leaf spot is a serious disease on St. Augustinegrass (Stenotaphrum secundatum), perennial ryegrass (Lolium perenne), and tall fescue (Festuca arundinacea). Virulence assays performed in this study revealed that M. grisea collected from rice could also cause disease on St. Augustinegrass and tall fescue. One rice isolate, Che86061, caused similar disease reactions on susceptible cultivars of rice and St. Augustinegrass and an incompatible interaction on resistant cultivars of both species. To explore whether similar defense-related genes are expressed in rice and St. Augustinegrass, a rice cDNA library was screened using pooled cDNAs derived from M. grisea-infected St. Augustinegrass. Thirty rice EST (expressed sequence tag) clones showing differential expression in St. Augustinegrass following M. grisea inoculation were identified and classified into six putative functional groups. Northern blot analyses of seven EST clones that collectively represented each putative functional group confirmed that the expression of five out of seven EST clones was similar in both rice and St. Augustinegrass. This study represents one of the first attempts to use a broad-scale genomic approach and resources of a model monocot system to study defense gene expression in St. Augustinegrass following M. grisea infection.

Inducible Overexpression of a Rice Allene Oxide Synthase Gene Increases the Endogenous Jasmonic Acid Level, PR Gene Expression, and Host Resistance to Fungal Infection

Many studies in dicotyledonous plants have shown that jasmonates, including jasmonic acid (JA) and methyl jasmonate, are important signal molecules involved in induced resistance to pathogen infection and insect herbivory. However, very little genetic and molecular evidence is available to demonstrate their role in host defense response of rice and other economically important monocot plants. In this study, we have shown that exogenous application of JA was able to activate defense gene expression and local induced resistance in rice seedlings against the rice blast fungus (Magnaporthe grisea). Furthermore, we have characterized a pathogen-inducible rice OsAOS2 gene (which encodes allene oxide synthase, a key enzyme in the JA biosynthetic pathway) and examined the role of endogenous JA in rice defense response through transgenic manipulation of the JA biosynthesis. Sequence analysis indicated that OsAOS2 contains four common domains of the cytochrome P450 enzyme, but does not have the signal peptide for chloroplast targeting. The basal level of OsAOS2 expression is very low in leaves but relatively high in the sheath, culm, and flower of rice plants. Interestingly, the expression of OsAOS2 in rice leaves can be induced significantly upon M. grisea infection. Transgenic rice lines carrying the OsAOS2 transgene under the control of a strong, pathogen-inducible PBZ1 promoter accumulated abundant OsAOS2 transcripts and higher levels of JA, especially after the pathogen infection. These transgenic lines also exhibited enhanced activation of pathogenesis-related (PR) genes such as PR1a, PR3, and PR5 and increased resistance to M. grisea infection. Our results suggest that JA plays a significant role in PR gene induction and blast resistance in rice plants.

Rice Defense Mechanisms AgainstCochliobolus miyabeanusandMagnaporthe griseaAre Distinct

ABSTRACT Responses of rice to Magnaporthe grisea and Cochliobolus miyabeanus were compared. In Tetep, a rice cultivar resistant to both fungi, pathogen inoculation rapidly triggered the hypersensitive reaction (HR), resulting in microscopic cell death. In rice cv. Nakdong, susceptible to both pathogens, M. grisea did not cause HR, whereas C. miyabeanus caused rapid cell death similar to that associated with HR, which appeared similar to that observed in cv. Tetep, yet failed to block fungal ramification. Treatment with conidial germination fluid (CGF) from C. miyabeanus induced rapid cell death in both cultivars, suggesting the presence of phytotoxins in CGF. Pretreatment of cv. Nakdong with CGF significantly increased resistance to M. grisea, while the same treatment was ineffective against C. miyabeanus. Similarly, in cv. Nakdong, benzothiadiazole (BTH) significantly increased resistance to M. grisea, but was ineffective against C. miyabeanus. Methyl jasmonate (MeJA) treatment appeared to be ineffective against either fungus. Increased resistance of cv. Nakdong to M. grisea by BTH or CCF treatment was correlated with more rapid induction of three monitored PR genes. Application of MeJA resulted in the expression of JAmyb in cv. Nakdong being induced faster than in untreated plants in response to M. grisea infection. In contrast, the expression pattern of the PR and JAmyb genes in response to C. miyabeanus was nearly identical between cvs. Nakdong and Tetep, and neither BTH nor MeJA treatment significantly modified their expression patterns in response to C. miyabeanus infection. Our results suggest that rice employs distinct mechanisms for its defense against M. grisea versus C. miyabeanus.